Thalidomide analogs and methods of use

ABSTRACT

Thalidomide analogs and methods of using the thalidomide analogs are disclosed. Some embodiments of the disclosed compounds exhibit anti-angiogenic and/or anti-inflammatory activity. Certain embodiments of the disclosed compounds are non-teratogenic.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/910,708, filed Jun. 24, 2020, which is a continuation of U.S.application Ser. No. 15/764,193, filed Mar. 28, 2018, now U.S. Pat. No.10,730,835, which is the U.S. National Stage of InternationalApplication No. PCT/US2016/054430, filed Sep. 29, 2016, which waspublished in English under PCT Article 21(2), which in turn claims thebenefit of U.S. Provisional Application No. 62/235,105, filed Sep. 30,2015, each of which is incorporated herein in its entirety.

FIELD

This disclosure concerns thalidomide analogs, particularlyidentification of non-teratogenic analogs, and methods of use.

BACKGROUND

Thalidomide (N-α-phthalimidoglutarimide) is an infamous drug known forits potent teratogenic side effects. Thalidomide was first synthesizedin Germany in 1954 and was marketed from 1957 worldwide as anon-barbiturate, non-addictive, non-toxic sedative and anti-nauseamedication. Thalidomide was withdrawn from the world market in 1961 dueto the development of severe congenital abnormalities in babies born tomothers using it for morning sickness. Thalidomide caused thousands ofcases of limb reduction anomalies, including phocomelia (absence of thelong bones in the forelimb) or amelia (a complete absence of theforelimb) in the children of pregnant women in the 1950s and 1960s.Other phenotypic malformations were also commonly seen including eye,ear, heart, gastrointestinal and kidney defects. Analogs of thalidomideare also commonly teratogenic.

Subsequent use, and research into the underlying mechanisms of action ofthalidomide, found it perpetuates inflammatory and immunomodulatorycharacteristics. The immunological and inflammatory basis for theclinical efficacy of thalidomide lies in thalidomide's ability toinhibit the synthesis of tumor necrosis factor alpha (TNF-α). TNF-α andfamily members play pivotal roles in a variety of physiological andpathological processes, which include cell proliferation anddifferentiation, apoptosis, the modulation of immune responses andinduction of inflammation. Thalidomide has also been shown to alter thedensity of TNF-α induced adhesion molecules on leukocytes in order toprevent the binding of the pro-inflammatory cytokine, which maycontribute to the anti-inflammatory properties of the drug.

Thalidomide has additionally been found to successfully inhibit theformation of new blood vessels, a process known as angiogenesis. Thisled to the hypothesis that a loss of normal blood vessel formation couldhave caused the damage seen in embryos following thalidomide exposure.Since then, thalidomide has been shown to inhibit new vessel formationin chicken embryos and also both the rat aortic ring assay, and in invitro cultures of human umbilical vein endothelial cells by modulationof the actin cytoskeleton. Thalidomide has been shown to treathereditary hemorrhagic telangiectasia in human patients by stabilizingleaky, malformed vessels and inhibiting their formation. Thalidomideanalogs have also been demonstrated to be anti-angiogenic in chickenembryo assays and zebrafish embryos. Thalidomide's combinedanti-angiogenic and anti-inflammatory properties likely lead to itsanti-cancer effects and efficacy in the treatment of multiple myeloma aswell as documented activity in other cancers.

A need exists for thalidomide analogs that exhibit clinical potential(e.g., anti-inflammatory and/or anti-angiogenic activity) withoutteratogenicity and/or that have more potency and/or fewer side effectsthan those of currently used thalidomide analogs.

SUMMARY

This disclosure concerns analogs of thalidomide, pharmaceuticallyacceptable salts thereof, and pharmaceutical compositions thereof.Methods of using the analogs are also disclosed.

A thalidomide analog has a structure according to general formula I or

where general formula I is

With respect to general formula I, Y¹ is a bond, —CH₂—, or —CH(CH₃)—. Ais —NH₃X where X is an anion with a −1 charge, or A is general formulaII

where bonds represented by “

” are optional bonds, and each bond represented by “

” is a single or double bond as needed to satisfy valence requirements;R¹ is —H, —NO₂, —NH₂, —OC(O)CH₃, or —NO₂H; R² is —H, —NH₂, or—N(H)CH(CH₃)₂; Z¹ is CH₂, C═O, or CH; and Z² is CH₂, C═O, C═S,

Ring B is:

where each bond represented by “

” is a single or double bond as needed to satisfy valence requirements;Z³ is C═O, C═S or CH; Z⁴ is C═O, C═S or CH, and at least one of Z³ andZ⁴ is C═O or C═S; R³ is —H or —OH; R⁴ is —H or —CH₃ and R⁵ and R⁶ areboth —H or both —CH₃.

In one embodiment, the thalidomide analog is non-teratogenic in azebrafish embryo assay and/or a chicken embryo assay at a concentrationwithin a range of 10-200 μg/mL. In an independent embodiment, thethalidomide analog possesses anti-inflammatory properties, but does notpossess anti-angiogenic properties. In another independent embodiment,the thalidomide analog possesses anti-angiogenic properties, but doesnot possess anti-inflammatory properties. In yet another independentembodiment, the thalidomide analog possesses anti-inflammatory andanti-angiogenic properties.

A pharmaceutical composition includes a thalidomide analog orpharmaceutically acceptable salt thereof as disclosed herein and atleast one pharmaceutically acceptable carrier or excipient.

A method for inhibiting TNF-α activity, TNF-α synthesis, angiogenesis,inflammation, or a combination thereof, includes contacting a cell withan effective amount of a thalidomide analog as disclosed herein or apharmaceutically acceptable salt thereof. In some embodiments, thethalidomide analog is non-teratogenic. In certain embodiments, thethalidomide analog is:

In certain embodiments, the thalidomide analog is compound 7, 9, 46, 59,65, 74, or 86.

In any or all of the above embodiments, contacting the cell with aneffective amount of the thalidomide analog may include administering toa subject a therapeutically effective amount of the thalidomide analogor pharmaceutically acceptable salt thereof or a therapeuticallyeffective amount of a pharmaceutical composition comprising thethalidomide analog or pharmaceutically acceptable salt thereof. In someembodiments, the subject is administered a second therapeutic agent,such as an anti-cancer agent, an anti-angiogenic agent, or ananti-inflammatory agent.

A method for inhibiting inflammation in a subject includes administeringto the subject a therapeutically effective amount of a thalidomideanalog as disclosed herein, wherein the thalidomide analog isnon-teratogenic and possesses anti-inflammatory properties. Thethalidomide analog may be compound 7, 9, 46, 59, 64, 65, 72, 74, 77, or86, as shown above. In certain embodiments, the thalidomide analog iscompound 7, 9, 46, 59, 65, 74, or 86. In some embodiments, thenon-teratogenic compound is administered orally, parenterally, rectally,nasally, buccally, vaginally, topically, optically, by inhalation spray,or via an implanted reservoir. In certain embodiments, the subject isadministered a second therapeutic agent, such as an anti-inflammatoryagent.

A method for treating an inflammatory disorder in a subject includesadministering to the subject a therapeutically effective amount of athalidomide analog as disclosed herein, wherein the thalidomide analogis non-teratogenic and possesses anti-inflammatory properties. In someembodiments, the non-teratogenic thalidomide analog is compound 7, 9,46, 59, 64, 65, 72, 74, 77, or 86. In certain embodiments, thethalidomide analog is compound 7, 9, 46, 59, 65, 74, or 86. In any orall of the foregoing embodiments, the inflammatory disorder may be aneurodegenerative disorder. In any or all of the foregoing embodiments,the subject may be administered a second therapeutic agent, such as ananti-inflammatory agent.

A method for inhibiting TNF-α activity, TNF-α synthesis, or acombination thereof in a subject includes administering to the subject atherapeutically effective amount of a non-teratogenic thalidomideanalog. In some embodiments, the non-teratogenic thalidomide analog iscompound 7, 9, 46, 59, 64, 65, 72, 74, 77, or 86. In certainembodiments, the thalidomide analog is compound 7, 9, 46, 59, 65, 74, or86.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary synthetic schemes for making substitutedphthalimides (thalidomide analogs).

FIG. 2 shows additional exemplary synthetic schemes for makingsubstituted phthalimidines.

FIGS. 3A-3L are photomicrographs of fil1:EGFP zebrafish after incubationwith vehicle or compounds for 3, 6, or 24 hours. The compounds wereadded at 24 hours post fertilization (hpf) and images were obtained at48 hpf (i.e., 24 hours post drug administration). In comparison tocontrol zebrafish (FIGS. 3A-3C) that show normal patterning of theintersegmental vessels (FIG. 3C, white arrow), treated and responsivezebrafish show a decrease in blood vessel length (FIG. 3F, white arrow)and loss of vascular connectivity or a decrease in the number of formingblood vessels (positive control CPS49, FIGS. 3D-3F). The examples shownare representative of treatment with compound 18 (50 μg/mL, FIGS. 3G-3I)and compound 4 (50 μg/mL, FIGS. 3J-3L).

FIG. 4 is a graph of vessel number versus vessel length demonstratingthe anti-angiogenic activity of each compound in fil1:EGFP zebrafishembryos.

FIGS. 5A-5D are photomicrographs of zebrafish embryos transgenic for afluorescently tagged neutrophil marker (Tg(mpo::GFP)) that were finclipped at 72 hpf to induce an inflammatory response. Arrows indicatethe edge of the cut; the scale bar is 100 μm. FIG. 5A is a control; FIG.5B is an embryo exposed to compound 51 (200 μg/mL); FIG. 5C is an embryoexposed to compound 20 (10 μg/mL); FIG. 5D is an embryo exposed tocompound 58 (10 μg/mL).

FIG. 6 is a graph quantifying the effects of thalidomide analogs on theinflammatory response in zebrafish embryos transgenic for afluorescently tagged neutrophil marker (Tg(mpo::GFP)) that were finclipped at 72 hpf to induce an inflammatory response. The scale barrepresents 100 μm.

FIGS. 7A-7P are photomicrographs showing defects seen in zebrafish andchicken embryos following thalidomide analog exposure. Embryos withexposure to a vehicle control showed normal development of the eye (e)(FIG. 7A) otic vesicle (o) (FIG. 7A), and pectoral fins (pf) (FIG. 7B).An anti-angiogenic compound caused microophthalmia (FIG. 7C, whitearrow) and malformation in fin development (FIG. 7D, white arrow). Acompound with no effect in the assays had no effect on development(FIGS. 7E, 7F). An anti-inflammatory compound had no effect on eye (FIG.7G) or fin (FIG. 7H) development. A chick embryo control in ovo hadnormal vasculature (FIG. 7I). Compound 23 caused constriction invasculature (white arrow) and necrosis (black arrow) of thechorioallantoic membrane (FIG. 7J). FIG. 7K is a control embryo ex ovo.An embryo treated with compound 80 exhibited microophthalmia (asterisk),limb reduction (white arrow), and hemorrhaging throughout the body (FIG.7L). FIG. 7M shows a normal eye. An embryo treated with compound 81exhibited growth reduction and hemorrhaging throughout the head (whitearrow) (FIG. 7N). FIG. 7O shows a control forelimb. An embryo treatedwith compound 11 had a limb reduction defect (asterisk, FIG. 7P, exovo).

FIGS. 8A and 8B are bar graphs showing effects of thalidomide analogs oncell viability, nitrite levels, and TNF-α protein levels atconcentrations ranging from 0-100 μM.

FIG. 9 is a graph showing microvessel outgrowth from rat aortic ringsincubated with thalidomide compounds for five days. Error bar representsthe standard error of the mean. Control n=11, TNP-470 n=7, thalidomideanalogs n=3.

FIGS. 10A and 10B are graphs showing inhibition of lattice outgrowth inhuman umbilical vein endothelial cells (HUVEC) by thalidomide analogs atconcentrations of 1 μM, 10 μM, and 30 μM. Error bar represents thestandard error of the mean; n=3.

FIG. 11 is a one-dose mean graph showing the effect of compound 29 oncell growth and cytotoxicity in 59 cancer cell lines.

FIG. 12 is a one-dose mean graph showing the effect of compound 86 oncell growth and cytotoxicity in 59 cancer cell lines.

FIGS. 13A-13E show anti-angiogenic activity of compounds 29 and 86 in ahuman-relevant ex vivo model—the human saphenous vein assay ofangiogenesis. FIG. 13A is a graph quantifying microvessel outgrowth incontrol, positive control, and thalidomide analog-treated humansaphenous vein sections. Error bar represents the standard error of themean. FIG. 13B is an image of outgrowth with no treatment; FIG. 13Cshows outgrowth inhibition by TNP-470; FIG. 13D shows no inhibition bycompound 29 at 10 μM concentration; FIG. 13E shows vastly reducedoutgrowth by compound 86 at 10 μM concentration.

FIG. 14 is graph monitoring weight loss as a measurement of toxicity inmice receiving thalidomide analogs. Error bar represents the standarderror of the mean (n=5 per treatment group).

FIG. 15 is a graph monitoring changes in tumor size in mice receivingthalidomide analogs.

DETAILED DESCRIPTION

Embodiments of thalidomide analogs are disclosed, as well as methods ofusing the analogs. Thalidomide is a tricyclic derivative of glutamicacid having the chemical structure:

Certain embodiments of the disclosed compounds were unexpectedly foundto be non-teratogenic.

I. Definitions

The following explanations of terms and abbreviations are provided tobetter describe the present disclosure and to guide those of ordinaryskill in the art in the practice of the present disclosure. As usedherein, “comprising” means “including” and the singular forms “a” or“an” or “the” include plural references unless the context clearlydictates otherwise. The term “or” refers to a single element of statedalternative elements or a combination of two or more elements, unlessthe context clearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features of thedisclosure are apparent from the following detailed description and theclaims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, percentages, temperatures, times, and soforth, as used in the specification or claims are to be understood asbeing modified by the term “about.” Accordingly, unless otherwiseindicated, implicitly or explicitly, the numerical parameters set forthare approximations that may depend on the desired properties soughtand/or limits of detection under standard test conditions/methods. Whendirectly and explicitly distinguishing embodiments from discussed priorart, the embodiment numbers are not approximates unless the word “about”is recited.

Definitions of common terms in chemistry may be found in Richard J.Lewis, Sr. (ed.), Hawley's Condensed Chemical Dictionary, published byJohn Wiley & Sons, Inc., 1997 (ISBN 0-471-29205-2). Definitions ofcommon terms in molecular biology may be found in Benjamin Lewin, GenesVII, published by Oxford University Press, 2000 (ISBN 019879276X);Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, publishedby Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by Wiley, John & Sons, Inc., 1995 (ISBN0471186341); and other similar references.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Angiogenesis: A physiological process through which new blood vesselsform from pre-existing vessels.

An anti-cancer agent is an agent that is used to treat malignancies.Exemplary anti-cancer agents include, but are not limited to,abiraterone, actinomycin D, altretamine, amifostine, anastrozole,asparaginase, bexarotene, bicalutamide, bleomycin, buserelin, busulfan,carboplatin, carmustine, chlorambucil cisplatin, cladribine, clodronate,combretastatin A4, cyclophosphamide, cyproterone, cytarabine,dacarbazine, daunorubicin, degarelix, diethylstilbestrol, docetaxel,doxorubicin, duocarmycin DM, epirubicin, ethinyl estradiol, etoposide,exemestane, 5-fluorouracil, fludarabine, flutamide, folinic acid,fulvestrant, gemcitabine, goserelin, ibandronic acid, idarubicin,ifosfamide, irinotecan, lanreotide, lenalidomide, letrozole,leuprorelin, medroxyprogesterone, megestrol, melphalan, mesna,methotrexate, octreotide, pamidronate, pemetrexed, mitocmycin, mitotane,mitoxantrone, oxaliplatin, paclitaxel, pentastatin, pipbroman,plicamycin, procarbazine, raltitrexed, stilbestrol, streptozocin,tamoxifen, temozolomide, teniposide, topotecan, triptorelin,vinblastine, vincristine, vinorelbine, and zolendronic acid.

Effective amount or therapeutically effective amount: An amountsufficient to provide a beneficial, or therapeutic, effect to a subjector a given percentage of subjects.

Excipient: A physiologically inert substance that is used as an additivein a pharmaceutical composition. As used herein, an excipient may beincorporated within particles of a pharmaceutical composition, or it maybe physically mixed with particles of a pharmaceutical composition. Anexcipient can be used, for example, to dilute an active agent and/or tomodify properties of a pharmaceutical composition. Examples ofexcipients include but are not limited to polyvinylpyrrolidone (PVP),tocopheryl polyethylene glycol 1000 succinate (also known as vitamin ETPGS, or TPGS), dipalmitoyl phosphatidyl choline (DPPC), trehalose,sodium bicarbonate, glycine, sodium citrate, and lactose.

Inflammation: A protective response to harmful stimuli, often elicitedby infection, irritation, injury or destruction of tissues. Inflammationcan be provoked by physical, chemical, and/or biologic agents.Inflammation involves immune cells, blood vessels, and molecularmediators such as vasoactive amines, plasma endopeptidases,prostaglandins, neutrophil products, lymphocyte factors, and others.Some hormones are anti-inflammatory while others are proinflammatory.Signs of inflammation include pain, heat, redness, swelling, and/or lossof function.

Pharmaceutically acceptable carrier: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington: The Science and Practice of Pharmacy, The University of theSciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins,Philadelphia, Pa., 21^(st) Edition (2005), describes compositions andformulations suitable for pharmaceutical delivery of one or morethalidomide analogs as disclosed herein.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. In some examples, the pharmaceutically acceptable carrier maybe sterile to be suitable for administration to a subject (for example,by parenteral, intramuscular, or subcutaneous injection). In addition tobiologically-neutral carriers, pharmaceutical compositions to beadministered can contain minor amounts of non-toxic auxiliarysubstances, such as wetting or emulsifying agents, preservatives, and pHbuffering agents and the like, for example sodium acetate or sorbitanmonolaurate.

Pharmaceutically acceptable salt: A biologically compatible salt of adisclosed compound, which salts are derived from a variety of organicand inorganic counter ions well known in the art and include, by way ofexample only, sodium, potassium, calcium, magnesium, ammonium,tetraalkylammonium, and the like; and when the molecule contains a basicfunctionality, salts of organic or inorganic acids, such ashydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate,oxalate, and the like. Pharmaceutically acceptable acid addition saltsare those salts that retain the biological effectiveness of the freebases while formed by acid partners that are not biologically orotherwise undesirable, e.g., inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and thelike, as well as organic acids such as acetic acid, trifluoroaceticacid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleicacid, malonic acid, succinic acid, fumaric acid, tartaric acid, citricacid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike. Pharmaceutically acceptable base addition salts include thosederived from inorganic bases such as sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. Exemplary salts are the ammonium, potassium, sodium,calcium, and magnesium salts. Salts derived from pharmaceuticallyacceptable organic non-toxic bases include, but are not limited to,salts of primary, secondary, and tertiary amines, substituted aminesincluding naturally occurring substituted amines, cyclic amines andbasic ion exchange resins, such as isopropylamine, trimethylamine,diethylamine, triethylamine, tripropylamine, ethanolamine,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, ethylenediamine, glucosamine, methylglucamine, theobromine,purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins,and the like. Exemplary organic bases are isopropylamine, diethylamine,ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.(See, for example, S. M. Berge, et al., “Pharmaceutical Salts,” J.Pharm. Sci., 1977; 66:1-19, which is incorporated herein by reference.)For therapeutic use, salts of the compounds are those wherein thecounter-ion is pharmaceutically acceptable. However, salts of acids andbases which are non-pharmaceutically acceptable may also find use, forexample, in the preparation or purification of a pharmaceuticallyacceptable compound.

Pharmaceutical composition: A composition that includes an amount (forexample, a unit dosage) of one or more of the disclosed compoundstogether with one or more non-toxic pharmaceutically acceptableadditives, including carriers, diluents, and/or adjuvants, andoptionally other biologically active ingredients. Such pharmaceuticalcompositions can be prepared by standard pharmaceutical formulationtechniques such as those disclosed in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. (19th Edition).

Stereoisomers: Isomers that differ in the arrangement of their atoms inspace are termed “stereoisomers”. Stereoisomers that are not mirrorimages of one another are termed “diastereomers” and those that arenon-superimposable mirror images of each other are termed “enantiomers.”When a compound has an asymmetric center, for example, if a carbon atomis bonded to four different groups, a pair of enantiomers is possible.An enantiomer can be characterized by the absolute configuration of itsasymmetric center and is described by the R- and S-sequencing rules ofCahn and Prelog, or by the manner in which the molecule rotates theplane of polarized light and designated as dextrorotatory orlevorotatory (i.e., as (+) or (−) isomers respectively). A chiralcompound can exist as either individual enantiomer or as a mixturethereof. A mixture containing equal proportions of the enantiomers iscalled a “racemic mixture.” E/Z isomers are isomers that differ in thestereochemistry of a double bond. An E isomer (from entgegen, the Germanword for “opposite”) has a trans-configuration at the double bond, inwhich the two groups of highest priority are on opposite sides of thedouble bond. A Z isomer (from zusammen, the German word for “together”)has a cis-configuration at the double bond, in which the two groups ofhighest priority are on the same side of the double bond. When spatialorientation is not indicated in a chemical formula, the formula includesall possible spatial orientations.

Teratogenic: Able to disturb the growth and/or development of an embryoor fetus, e.g., able to cause physical defects. A teratogen orteratogenic agent is an agent that induces or increases incidence ofabnormal prenatal development. Exemplary teratogens include certainviruses, drugs, or radiation that cause malformations or functionaldamage to an embryo or fetus. A non-teratogenic agent or compound doesnot have adverse effects on the growth and/or development of an embryoor fetus.

Treat(ing) or treatment: With respect to a disease or disorder, eitherterm includes (1) preventing the disease or disorder, e.g., causing theclinical symptoms of the disease or disorder not to develop in an animalthat may be exposed to or predisposed to the disease or disorder butdoes not yet experience or display symptoms of the disease or disorder,(2) inhibiting the disease or disorder, e.g., arresting the developmentof the disease or disorder or its clinical symptoms, and/or (3)relieving the disease or disorder, e.g., causing regression of thedisease or disorder or its clinical symptoms.

II. Thalidomide Analogs and Pharmaceutical Compositions

This disclosure concerns thalidomide analogs. Some embodiments of thedisclosed compounds exhibit anti-angiogenic properties and/oranti-inflammatory properties, and as such can be used to treat a widevariety of pathological conditions that are linked to angiogenesisand/or inflammation. Certain embodiments of the disclosed compoundsmodulate TNF-α activity, TNF-α synthesis, and/or angiogenesis. Thecompounds also may inhibit inducible nitric oxide synthase (iNOS) andproinflammatory cytokines such as IFN-γ, IL-2 and IL-17.Pharmaceutically acceptable salts, stereoisomers, and metabolites of allof the disclosed compounds also are contemplated. In some embodiments,the compounds are lenalidomide or pomalidomide derivatives in whichcarbonyl groups in corresponding non-sulfur-containing lenalidomide orpomalidomide derivatives are replaced by one or more thiocarbonylgroups.

Most embodiments of the disclosed compounds have a structure accordingto general formula I:

where Y¹ is a bond, —CH₂—, or —CH(CH₃)—. A is —NH₃X where X is an anionwith a −1 charge, or A is general formula II:

where bonds represented by “

” are optional bonds, and each bond represented by “

” is a single or double bond as needed to satisfy valence requirements.R¹ is —H, —NO₂, —NH₂, —OC(O)CH₃, or —NO₂H; and R² is —H, —NH₂, or—N(H)CH(CH₃)₂. In some embodiments at least one of R¹ and R² is —H. Z¹is CH₂, C═O, or CH; and Z² is CH₂, C═O, C═S,

Ring B is:

where each bond represented by “

” is a single or double bond as needed to satisfy valence requirements.Z³ is C═O, C═S, or CH; Z⁴ is C═O, C═S, or CH; and at least one of Z³ andZ⁴ is C═O or C═S. R³ is —H or —OH, R⁴ is —H or —CH₃, and R⁵ and R⁶ areboth —H or both —CH₃.

In some embodiments, the following provisos apply.

When ring B is

then: (i) R¹ is not —NH₂; (ii) if R¹ is —NO₂ and Y¹ is a bond, then atleast one of Z¹ and Z² is C═O and one of Z³ and Z⁴ is other than C═O orC═S, or if both Z¹ and Z² are C═O, then Z³ is C═O or C═S and Z⁴ is C═S;(iii) if R² is —NH₂ and Y¹ is a bond, then one of Z¹ and Z² is otherthan C═O, and one of Z³ and Z⁴ is other than C═O or C═S; (iv) if R² is—NO₂ and Y¹ is a bond, then one of Z³ and Z⁴ is other than C═O or C═S;(v) if R² is N(H)CH(CH₃)₂, Y¹ is a bond, Z¹ is CH₂ and Z² is C═O, thenone of Z³ and Z⁴ is other than C═O; (vi) if A is —NH₃X, X is CF₃CO₂, andY¹ is a bond, then at least one of Z³ and Z⁴ is other than C═O.

When ring B is

R¹ is —H, —NH₂ or —NO₂, R² and R³ are —H, Z¹ is CH₂, and Z² is C═O, thenY¹ is not a bond.

When ring B is

R¹ and R² are H, Z¹ is CH₂ and Z² is C═O, then Y¹ is not a bond.

When ring B is

and R¹ and R² are H, then one of Z¹ and Z² is other than C═O.

In some embodiments, when ring B is

then A is

In certain embodiments, A is

In some embodiments, when ring B is

the stereochemistry is

In some embodiments where R⁴-R⁶ are H, the stereochemistry is

In some embodiments, when ring B is

the stereochemistry is

In some embodiments, when ring B is

the stereochemistry is

In an independent embodiment, the disclosed analog is

In some embodiments, A is

and ring B is:

Exemplary compounds are shown in Groups I-VI:

Some embodiments of the disclosed compounds possess anti-inflammatoryand/or anti-angiogenic properties. It was unexpectedly discovered thatcertain of the disclosed compounds are non-teratogenic. In someembodiments, the thalidomide analog is non-teratogenic in a zebrafishembryo assay and/or a chicken embryo assay at a range of concentrationsand developmental time points. In some examples, the thalidomide analogwas non-teratogenic in the zebrafish embryo assay and/or chicken embryoassay at a concentration within a range of 10-200 μg/mL. It isunderstood that concentrations at which the thalidomide analog isnon-teratogenic may vary in other animal models. In some embodiments,the thalidomide analog is non-teratogenic at a concentration of 0.001 mMto 10 mM, such as from 0.01-1 mM or from 0.02-0.5 mM. In certainembodiments, the thalidomide analog is non-teratogenic at atherapeutically effective dose. Advantageously, the thalidomide analogmay be non-neurotoxic at a therapeutically effective dose. In oneindependent embodiment, the thalidomide analog possessesanti-inflammatory properties and is non-teratogenic. In anotherindependent embodiment, the thalidomide analog possessesanti-inflammatory properties, does not possess anti-angiogenicproperties, and is non-teratogenic. In yet another independentembodiment, the thalidomide analog possesses anti-inflammatoryproperties and is non-teratogenic and non-neurotoxic. Exemplarynon-teratogenic compounds are shown in Table 1:

7

9

46

59

64

65

72

74

77

86In some embodiments, the non-teratogenic analog is compound 7, 9, 46,59, 65, 74, or 86.

The disclosed compounds can be combined with pharmaceutically acceptablecarriers, excipients, and optionally sustained-release matrices, such asbiodegradable polymers, to form therapeutic compositions. Therefore,also disclosed are pharmaceutical compositions including one or more ofany of the compounds disclosed above, a pharmaceutically acceptablecarrier and/or excipient. The composition may comprise a unit dosageform of the composition, and may further comprise instructions foradministering the composition to a subject to inhibit angiogenesis, forexample, instructions for administering the composition to achieve ananti-tumor effect or to inhibit a pathological angiogenesis. Suchpharmaceutical compositions may be used in methods for modulatingangiogenesis, inflammation, TNF-α activity, and/or TNF-α synthesis in asubject by administering to the subject a therapeutically effectiveamount of the composition.

The disclosed pharmaceutical compositions can be in the form of tablets,capsules, powders, granules, lozenges, liquid or gel preparations, suchas oral, topical, or sterile parenteral solutions or suspensions (e.g.,eye or ear drops, throat or nasal sprays, etc.), transdermal patches,and other forms known in the art.

Pharmaceutical compositions can be administered systemically or locallyin any manner appropriate to the treatment of a given condition,including orally, parenterally, rectally, nasally, buccally, vaginally,topically, optically, by inhalation spray, or via an implantedreservoir. The term “parenterally” as used herein includes, but is notlimited to subcutaneous, intravenous, intramuscular, intrasternal,intrasynovial, intrathecal, intrahepatic, intralesional, andintracranial administration, for example, by injection or infusion. Fortreatment of the central nervous system, the pharmaceutical compositionsmay readily penetrate the blood-brain barrier when peripherally orintraventricularly administered.

Pharmaceutically acceptable carriers include, but are not limited to,ion exchangers, alumina, aluminum stearate, lecithin, serum proteins(such as human serum albumin), buffers (such as phosphates), glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol, andwool fat.

Tablets and capsules for oral administration can be in a form suitablefor unit dose presentation and can contain conventional pharmaceuticallyacceptable excipients. Examples of these include binding agents such assyrup, acacia, gelatin, sorbitol, tragacanth, and polyvinylpyrrolidone;fillers such as lactose, sugar, corn starch, calcium phosphate,sorbitol, or glycine; tableting lubricants, such as magnesium stearate,talc, polyethylene glycol, or silica; disintegrants, such as potatostarch; and dispersing or wetting agents, such as sodium lauryl sulfate.Oral liquid preparations can be in the form of, for example, aqueous oroily suspensions, solutions, emulsions, syrups or elixirs, or can bepresented as a dry product for reconstitution with water or othersuitable vehicle before use.

The pharmaceutical compositions can also be administered parenterally ina sterile aqueous or oleaginous medium. The composition can be dissolvedor suspended in a non-toxic parenterally-acceptable diluent or solvent,e.g., as a solution in 1,3-butanediol. Commonly used vehicles andsolvents include water, physiological saline, Hank's solution, Ringer'ssolution, and sterile, fixed oils, including synthetic mono- ordi-glycerides, etc. For topical application, the drug may be made upinto a solution, suspension, cream, lotion, or ointment in a suitableaqueous or non-aqueous vehicle. Additives may also be included, forexample, buffers such as sodium metabisulphite or disodium edetate;preservatives such as bactericidal and fungicidal agents, includingphenyl mercuric acetate or nitrate, benzalkonium chloride orchlorhexidine, and thickening agents, such as hypromellose.

The compounds can be used in the form of pharmaceutically acceptablesalts derived from inorganic or organic acids and bases, including, butnot limited to: acetate, adipate, alginate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, citrate, camphorate,camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate.Base salts include, but are not limited to, ammonium salts, alkali metalsalts (such as sodium and potassium salts), alkaline earth metal salts(such as calcium and magnesium salts), salts with organic bases (such asdicyclohexylamine salts), N-methyl-D-glucamine, and salts with aminoacids (such as arginine, lysine, etc.). Basic nitrogen-containing groupscan be quaternized, for example, with such agents as C1-8 alkyl halides(such as methyl, ethyl, propyl, and butyl chlorides, bromides, andiodides), dialkyl sulfates (such as dimethyl, diethyl, dibutyl, anddiamyl sulfates), long-chain halides (such as decyl, lauryl, myristyl,and stearyl chlorides, bromides, and iodides), aralkyl halides (such asbenzyl and phenethyl bromides), etc. Water or oil-soluble or dispersibleproducts are produced thereby.

III. Uses and Methods of Use

The compounds disclosed herein, and pharmaceutically acceptable saltsthereof, may be used for inhibiting TNF-α activity, TNF-α synthesis,angiogenesis, inflammation, or a combination thereof. In one embodiment,a cell is contacted with an effective amount of a thalidomide analog asdisclosed herein, to inhibit TNF-α activity, TNF-α synthesis,angiogenesis, inflammation, or a combination thereof. The cell may becontacted in vitro, in vivo, or ex vivo. In one embodiment, the cell iscontacted with a thalidomide analog in Groups I-VI. In anotherembodiment, the cell is contacted with a thalidomide analog in Table 1.In an independent embodiment, the cell is contacted with compound 7, 9,46, 59, 65, 74, or 86. In any of the foregoing embodiments, contactingthe cell with an effective amount of the thalidomide analog may compriseadministering to a subject a therapeutically effective amount of thethalidomide analog or pharmaceutically acceptable salt thereof or atherapeutically effective amount of a pharmaceutical compositioncomprising the thalidomide analog or pharmaceutically acceptable saltthereof. Administration may be performed by any suitable route,including orally, parenterally, rectally, nasally, buccally, vaginally,topically, optically, by inhalation spray, or via an implantedreservoir.

In one embodiment, a subject is administered a therapeutically effectiveamount of a thalidomide analog according to general formula I, apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition comprising the compound. In another embodiment, a subject isadministered a therapeutically effective amount of a thalidomide analogin Groups I-VI, a pharmaceutically acceptable salt thereof, or apharmaceutical composition comprising the compound. In yet anotherembodiment, a subject is administered a therapeutically effective amountof a thalidomide analog in Table 1, a pharmaceutically acceptable saltthereof, or a pharmaceutical composition thereof. In certainembodiments, the thalidomide analog is compound 7, 9, 46, 59, 65, 74, or86. In any of the foregoing embodiments, the subject may have a disordermediated by TNF-α, angiogenesis and/or inflammation. Advantageously, thethalidomide analog in any of the foregoing embodiments may benon-teratogenic. The thalidomide analog also may be non-neurotoxic. Inparticular, the thalidomide analog may be non-teratogenic at atherapeutically effective dose, such as a therapeutically effectiveanti-inflammatory dose, a therapeutically effective anti-angiogenicdose, or both. In some embodiments, the thalidomide analog possessesanti-inflammatory properties. In certain embodiments, the thalidomideanalog does not possess anti-angiogenic properties. The compound isadministered to the subject by any suitable route, including orally,parenterally, rectally, nasally, buccally, vaginally, topically,optically, by inhalation spray, or via an implanted reservoir.

Some embodiments of the thalidomide analogs may be used for treatinginflammatory disorders and/or autoimmune disorders. In certainembodiments, a non-teratogenic thalidomide analog as disclosed herein isadministered to a subject to inhibit inflammation and/or treat adisorder mediated by inflammation. The non-teratogenic thalidomideanalog may possess anti-inflammatory properties while not possessinganti-angiogenic properties. Exemplary inflammatory and/or autoimmunedisorders that may be ameliorated with embodiments of the disclosedthalidomide analogs include, but are not limited to, rheumatoidarthritis, immune arthritis, degenerative arthritis, celiac disease,glomerulonephritis, lupus nephritis, prostatitis, inflammatory boweldisease (e.g., Crohn's disease), pelvic inflammatory disease,graft-versus-host disease, interstitial cystitis, autoimmunethyroiditis, Graves' disease; autoimmune pancreatitis, Sjogren'ssyndrome, myocarditis, autoimmune hepatitis, primary biliary cirrhosis,autoimmune angioedema, bullous pemphigoid, discoid lupus erythematosus,erythema nodosum leprosum, sarcoidosis, pemphigus vulgaris psoriasis,POEMS syndrome, polymyositis, human immune deficiency virus/acquiredimmune deficiency syndrome, vasculitis, and sarcopenia. Subject toneurotoxicity considerations (e.g., whether the analog is well toleratedby nervous tissue), certain embodiments of the thalidomide analogsdisclosed herein may be used to reduce neuroinflammation as a treatmentstrategy for neurodegenerative disorders. Advantageously, a thalidomideanalog used to reduce neuroinflammation may be non-neurotoxic at atherapeutically effective dose. Examples of neurodegenerative and/orneuroinflammatory disorders that may be ameliorated with embodiments ofthe disclosed thalidomide analogs include, but are not limited to,neurodegeneration resulting from head trauma (e.g., traumatic braininjury), spinal cord injuries, stroke, Alzheimer's disease, Parkinson'sdisease, ALS (amyotrophic lateral sclerosis), HIV (humanimmunodeficiency virus) dementia, Huntington's disease, multiplesclerosis, cerebral amyloid angiopathy, tauopathies, peripheralneuropathies, macular degeneration, hearing loss, cochlear injury,epilepsy, a non-epileptic seizure disorder (e.g., due to head injury,dementia, prenatal brain injury, meningitis, lupus, encephalitis, amongothers), and major depressive disorder (also known as clinicaldepression, unipolar depression). Embodiments of the disclosedthalidomide analogs may be used to reduce chronic systemic and CNSinflammation and/or as immunomodulatory agents. In some embodiments, thethalidomide analog is used to delay the onset of and/or progression ofsarcopenia. Embodiments of the disclosed thalidomide analogs are smallmolecular weight lipophilic compounds with physicochemical properties topass through the blood-brain barrier.

Evidence from clinical and preclinical studies indicates that basalinflammatory status increases as a function of normal aging, andprogressive development of a mild pro-inflammatory state closelyassociates with the major degenerative diseases of the elderly (Holmeset al., Neurology 73:768-74, 2009; Heneka et al., Lancet Neurol14:388-405, 2015). Hallmarks of aging include increased oxidative stressgenerated by reactive nitrogen and oxygen species, lipid peroxidation,and mitochondrial and DNA damage, particularly within the brain.Microarray studies indicate an overall rise in inflammatory andpro-oxidant genes with a decline in growth, anti-inflammatory andanti-oxidant genes in the brain as well as other key organs of olderversus adult rodents (Cribbs et al., J Neuroinflammation 9:179, 2012).In line with this, levels of brain pro-inflammatory cytokines have beenfound elevated with age in rodents and humans, and several regulatorymolecules and anti-inflammatory cytokines were reduced (Deleidi et al.,Front Neurosci 9:172, 2015). As a source of these pro- andanti-inflammatory molecules, microglia (representing some 15% of cellsin brain) are thereby implicated as the major culprit of thisneuroinflammation. Correcting the overproduction of pro-inflammatorycytokines generated by microglia provides a strategy to mitigate a broadnumber of neurodegenerative and systemic disorders in which aninflammatory component drives the disease process. Tumor necrosisfactor-α (TNF-α) is one of the primary pro-inflammatory cytokinessynthesized and released by microglial cells within the brain and byperipheral blood mononuclear cells PBMCs)/macrophages systemically. OnceTNF-α is released, it may initiate a self-propagating cycle of uncheckedinflammation (Frankola et al., CNS Neurol Disord Drug Targets10:391-403, 2011). Pharmacological intervention to interrupt this cyclemay be of significant benefit in the setting of inflammation-mediateddiseases. Reactive nitrogen and oxygen species are both regulators andeffectors of inflammation—by generation of nitric oxide (as followed byevaluating nitrite levels: its stable end product) andsuperoxide/peroxynitrite levels, and their levels can likewise bypharmacologically modulated/reduced by targeting TNF-α, e.g., byadministration of a thalidomide analog as disclosed herein.

Proinflammatory TNF-α released by microglia in brain and PBMCssystemically can, if not appropriately time-dependently regulated,initiate a self-propagating cycle of unchecked inflammation and has beenimplicated in the pathogenesis of a wide number of chronic (AD,Parkinson's disease, ALS) and acute (stroke, traumatic brain injury(TBI)) neurodegenerative disorders as well as autoimmune conditions(Clark & Vissel, J Neuroinflammation. 13:236, 2016; Clark & Vissel,Neural Plast. 2015:358263, 2015; Chatzantoni & Mouzaki, Curr Top MedChem. 6:1707-14, 2006). Across these disorders, TNF-α levels are foundconsistently elevated in biological fluids of animal models (Frankola etal., CNS Neurol Disord Drug Targets 10:391-403, 2011). In AD, forexample, by as much as 25-fold (Tarkowski E, et al. J Clin Immunol19:223-30, 1999). TNF-α by interacting via NF-κB not only induces theproduction of other proinflammatory cytokines/chemokines but feeds backto increase its own generation. Studies in subjects with mild cognitiveimpairment that progress to develop AD suggest that increased CSF TNF-αlevels are an early event, and their rise correlates with diseaseprogression (Tarkowski et al., J Neurol Neurosurg Psychiatry 74:1200-5,2003). Paralleling this, elevated expression of TNF-α transcripts arereported within the entorhinal cortex of transgenic mouse models of ADat 2 months, prior to any appearance of amyloid and tau pathology(Janelsins et al., J Neuroinflamm 2:23, 2005), and this increaseassociates with the onset of cognitive deficits in these mice (Billingset al., Neuron 45:675-88, 2005) and later neuronal loss. Likewise, inboth stroke and TBI, elevations in TNF-α are evident early in biologicalfluids and precede neuronal apoptosis (Yoon et al., J Neurosci Res.91:671-80, 2013; Chiu C C, J Neurosci Methods. 2016).

A method for inhibiting TNF-α activity and/or TNF-α synthesis in asubject using the disclosed thalidomide analogs is provided. The methodincludes administering a therapeutically effective amount of a disclosedthalidomide analog to a subject to achieve a TNF-α inhibitory effect.The disclosed thalidomide analogs having TNF-α inhibitory effects areuseful for treating many inflammatory, infectious, immunological, andmalignant diseases. These include but are not limited to septic shock,sepsis, endotoxic shock, hemodynamic shock and sepsis syndrome, postischemic reperfusion injury, malaria, mycobacterial infection,meningitis, psoriasis and other dermal diseases, congestive heartfailure, fibrotic disease, cachexia, graft rejection, cancer, tumorgrowth, undesirable angiogenesis, autoimmune disease, opportunisticinfections in AIDS, rheumatoid arthritis, rheumatoid spondylitis,osteoarthritis, other arthritic conditions, inflammatory bowel disease,Crohn's disease, ulcerative colitis, multiple sclerosis, systemic lupuserythematosus, ENL in leprosy, radiation damage, and hyperoxic alveolarinjury.

Still further, a method for modulating angiogenesis in a subject isprovided. The method includes administering to the subject atherapeutically effective amount of one or more of any of the disclosedthalidomide analogs. Desirably, the thalidomide analog possessesanti-angiogenic properties. In some embodiments, where ananti-angiogenic thalidomide analog is utilized, the therapeuticallyeffective amount of the compound can be administered to a subject with atumor to achieve an anti-tumor effect, such as inhibition oftumorigenesis or tumor metastasis. In other embodiments, thetherapeutically effective amount of the thalidomide analog isadministered to a subject with a pathological angiogenesis. In stillother embodiments, the therapeutically effective amount of thethalidomide analog is administered to a subject with anangiogenesis-mediated retinopathy, such as a non-cancerous retinopathy.

As angiogenesis inhibitors, some embodiments of the disclosedthalidomide analogs are useful in the treatment of both primary andmetastatic solid tumors, including carcinomas of breast, colon, rectum,lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver,gallbladder and bile ducts, small intestine, urinary tract (includingkidney, bladder and urothelium), female genital tract, (includingcervix, uterus, and ovaries as well as choriocarcinoma and gestationaltrophoblastic disease), male genital tract (including prostate, seminalvesicles, testes and germ cell tumors), endocrine glands (including thethyroid, adrenal, and pituitary glands), and skin, as well ashemangiomas, melanomas, sarcomas (including those arising from bone andsoft tissues as well as Kaposi's sarcoma) and tumors of the brain,nerves, eyes, and meninges (including astrocytomas, gliomas,glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas,and meningiomas). Such thalidomide analogs may also be useful intreating solid tumors arising from hematopoietic malignancies such asleukemias (i.e. chloromas, plasmacytomas and the plaques and tumors ofmycosis fungoides and cutaneous T-cell lymphoma/leukemia) as well as inthe treatment of lymphomas (both Hodgkin's and non-Hodgkin's lymphomas).In addition, these thalidomide analogs may be useful in the preventionof metastases from the tumors described above either when used alone orin combination with radiotherapy and/or other chemotherapeutic agents.The thalidomide analogs are also useful in treating multiple myeloma.

Embodiments of the disclosed thalidomide analogs with anti-angiogenicproperties can also be used to treat a pathological (i.e. abnormal,harmful or undesired) angiogenesis, for example, various ocular diseasessuch as diabetic retinopathy, retinopathy of prematurity, corneal graftrejection, retrolental fibroplasia, neovascular glaucoma, rubeosis,retinal neovascularization due to macular degeneration, hypoxia,angiogenesis in the eye associated with infection or surgicalintervention, and other abnormal neovascularization conditions of theeye; skin diseases such as psoriasis; blood vessel diseases such ashemangiomas, and capillary proliferation within atherosclerotic plaques;Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;and wound granulation. Other uses include the treatment of diseasescharacterized by excessive or abnormal stimulation of endothelial cells,including but not limited to intestinal adhesions, Crohn's disease,atherosclerosis, scleroderma, and hypertrophic scars, such as keloids.The disclosed thalidomide analogs are also useful in the treatment ofdiseases that have angiogenesis as a pathologic consequence such as catscratch disease (Rochele minalia quintosa) and ulcers (Helicobacterpylori). The disclosed thalidomide analogs are also useful to reducebleeding by administration prior to surgery, especially for thetreatment of resectable tumors.

In certain embodiments, the thalidomide analogs disclosed herein mayexhibit no toxicity or tolerable toxicity at dosages of up to 25mg/daily, such as up to 50 mg/daily or even up to 75 mg/daily. Thetherapeutically effective amount or amount depends, for example, on thecondition treated, nature of the formulation, nature of the condition,embodiment of the claimed pharmaceutical compositions, mode ofadministration, and condition and weight of the patient. Dosage levelsare typically sufficient to achieve a tissue concentration at the siteof action that is at least the same as a concentration that has beenshown to be active in vitro, in vivo, or in tissue culture. For example,a dosage of about 0.1 μg/kg body weight/day to about 1000 mg/kg bodyweight/day, for example, a dosage of about 1 μg/kg body weight/day toabout 1000 μg/kg body weight/day, such as a dosage of about 5 μg/kg bodyweight/day to about 500 μg/kg body weight/day can be useful fortreatment of a particular condition. Advantageously, certain embodimentsof the disclosed thalidomide analogs are non-teratogenic attherapeutically effective amounts.

The disclosed thalidomide analogs can be used in combination with othercompositions and procedures for the treatment of diseases. In someembodiments, the disclosed thalidomide analogs are used in combinationwith a second therapeutic agent, such as an anti-cancer agent, ananti-angiogenic agent, or an anti-inflammatory agent. For example, atumor can be treated conventionally with surgery, radiation orchemotherapy in combination with an anti-angiogeniccompound/concentration and then, optionally the compound/concentrationcan be further administered to the subject to extend the dormancy ofmicrometastases and to stabilize and inhibit the growth of any residualprimary tumor. Alternatively, an angiogenic compound or angiogenicconcentration of a compound can be used in combination with otherangiogenesis stimulating agents. For example, thermal energy (in theform of resistive heating, laser energy or both) to create thermallytreated stimulation zones or pockets (optionally interconnected, atleast initially, by small channels) in the tissue for the introductionof blood born growth and healing factors, along with stimulatedcapillary growth surrounding the thermally treated zones. Suchstimulation zones allow increased blood flow to previously ischemicand/or nonfunctional tissue (such as cardiac tissue) with a concomitantincreased supply of oxygen and nutrients ultimately resulting in arevitalization of the treated sections the tissue when used incombination with the angiogenic compositions/concentrations. In otherembodiments, disclosed thalidomide analogs exhibiting TNF-α inhibitoryactivity can be combined with other TNF-α inhibitory agents, forexample, steroids such as dexamethasone and prednisolone. When used fortreatment of a cancer, the thalidomide analogs can be used incombination with chemotherapeutic agents and/or radiation and/orsurgery.

Examples of other chemotherapeutic agents that can be used incombination with the disclosed thalidomide analogs include alkylatingagents, antimetabolites, natural products, kinase inhibitors, hormonesand their antagonists, and miscellaneous other agents. Examples ofalkylating agents include nitrogen mustards (such as mechlorethamine,cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkylsulfonates (such as busulfan), and nitrosoureas (such as carmustine,lomustine, semustine, streptozocin, or dacarbazine). Examples ofantimetabolites include folic acid analogs (such as methotrexate),pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs,such as mercaptopurine or thioguanine. Examples of natural productsinclude vinca alkaloids (such as vinblastine, vincristine, orvindesine), epipodophyllotoxins (such as etoposide or teniposide),antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin,plicamycin, or mitocycin C), and enzymes (such as L-asparaginase).Examples of kinase inhibitors include small molecule inhibitors (such asIressa, Tarceva, PKI-166, CI-1033, CGP-5923A, EKB-569, TAK165,GE-572016, CI-1033, SU5416, ZD4190, PTK787/ZK222584, CGP41251, CEP-5214,ZD6474, BIBF1000, VGA1102, SU6668, SU11248, CGP-57148, tricyclicquinoxalines, SU4984, SU5406, Gleevec, NSC680410, PD166326, PD1173952,CT53518, GTP14564, PKC412, PP1, PD116285, CGP77675, CGP76030, CEP-701,and CEP2583), ligand modulators (such as Bevacizumanb, MV833, SolubleFlt-1 and Flk-1, VEGF Trap, GFB 116, NM3, VEGF 121-diptheria toxinconjugate and Interfereon-α), and monoclonal antibodies againstreceptors (such as Cetuximab, ABX-EGF, Y10, MDX-447, h-R3, EMD 72000,herceptin, MDX-H210, pertuzumab, IMC-1C11, and MF1). Examples ofhormones and antagonists include adrenocorticosteroids (such asprednisone), progestins (such as hydroxyprogesterone caproate,medroxyprogesterone acdtate, and magestrol acetate), estrogens (such asdiethylstilbestrol and ethinyl estradiol), antiestrogens (such astamoxifen), and androgens (such as testerone proprionate andfluoxymesterone). Examples of miscellaneous agents include platinumcoordination complexes (such as cis-diamine-dichloroplatinum II, whichis also known as cisplatin), substituted ureas (such as hydroxyurea),methyl hydrazine derivatives (such as procarbazine), vaccines (such asAPC8024), AP22408, B43-genistein conjugate, paclitaxel, AG538, andadrenocrotical suppressants (such as mitotane and aminoglutethimide). Inaddition, the disclosed thalidomide analogs can be combined with genetherapy approaches, such as those targeting VEGF/VEGFR (includingantisense oligonucleotide therapy, Adenovirus-based Flt-1 gene therapy,Retrovirus-base Flk-1 gene therapy, Retrovirus-based VHL gene therapy,and angiozyme) and IGF-1R (including INX-4437). Examples of the mostcommonly used chemotherapy drugs that can be used in combination withthe disclosed tricyclic compounds agent include Adriamycin, Alkeran,Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan,Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide,Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard,Taxol, Velban, Vincristine, VP-16, Gemcitabine (Gemzar), Herceptin,Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571,Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin andcalcitriol.

The disclosed thalidomide analogs also can be combined with radiotherapyemploying radioisotopes (such as ³²P, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁷⁷Lu),particle beams (such as proton, neutron and electron beams) andelectromagnetic radiation (such as gamma rays, x-rays and photodynamictherapy using photosensitizers and visible or ultraviolet rays).

In some embodiments, the second therapeutic agent is ananti-inflammatory agent. Exemplary anti-inflammatory agents includenon-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen,ketoprofen, piroxicam, naproxen, sulindac, aspirin, cholinesubsalicylate, diflunisal, fenoprofen, indomethacin, meclofenamate,salsalate, tolmetin, ketorolac, flurbiprofen, and magnesium salicylate.

IV. Kits

Kits are also a feature of this disclosure. Embodiments of the kitsinclude at least one thalidomide analog as disclosed herein. In someembodiments, the kits also include at least one solution in which thethalidomide analog may be dissolved or suspended. In one embodiment, thesolution is suitable for directly dissolving or suspending thethalidomide analog. In an independent embodiment, the solution isprovided as a concentrated solution, which is subsequently diluted priorto use. The solution may be a pharmaceutically acceptable carrier. Thekits also may include one or more containers, such as a disposable testtube or cuvette. In certain embodiments, the thalidomide analog ispremeasured into one or more containers (e.g., test tubes, cuvettes, orampules). The kits may further include instructions for using thethalidomide analog.

V. Examples

Thalidomide analogs disclosed herein have not yet undergone regulatoryreview for use in humans

Materials and Methods:

Zebrafish embryology: The use of zebrafish embryo (Danio rerio)bioassays as a preclinical model in the classification of novelcompounds has increased in recent years due to several factorsincluding; low cost, ease of maintenance, speed of embryonicdevelopment, transparency of the embryos and genetic conservationbetween zebrafish and humans (Howe et al., Nature 2013; 496:498;Therapontos et al., PNAS 2009, 106:8573; Mahony et al., PNAS 2013,110:12703; Beedie et al., Oncotarget 2016, 7(22):33237; Beedie et al.,Mol. Cancer Ther. 2015, 14(10):2228). Two established zebrafish reporterlines were used—the fli1:EGFP line, where the fli1 promoter drivesexpression of enhanced green fluorescent protein within the developingvasculature (Lawson et al., 2002), and the Tg(mpo::EGFP) line (alsoknown as mpo:GFP), which expresses green fluorescent protein via theneutrophil specific myeloperoxidase promoter (Renshaw et al., Blood2006, 108:3976). The use of these zebrafish transgenic reporter linesallows for the identification of potential anti-angiogenic andanti-inflammatory compounds respectively, and both have been usedpreviously to successfully classify the action of thalidomide analogs(Therapontos et al., 2009; Mahony et al., 2013; Beedie et al.,Oncotarget 2016, 7(22):33237; Beedie et al., Mol. Cancer Ther. 2015,14(10):2228). Zebrafish embryos were treated with analogs as previouslydescribed (Mahony et al., 2013). Briefly, fli1:EGFP embryos werecollected and embryos were allowed to develop for 24 hours. Embryos weredechorionated manually and exposed to test compounds or vehicle controlfor a further 24 hours, imaged and analyzed for intersegmental vessel(ISV) growth. At 24 hours post fertilisation (hpf) the ISVs were idealto study to visualize and analyze the actions of compounds onangiogenesis. At this time point ISVs were rapidly forming beneath thedeveloping head, over the body and had yet to form in the tail (Lawsonand Weinstein, Developmental Biology 2002, 248:307; Therapontos andVargesson, Developmental Dynamics 2010, 239:2761). Additionally, thesesprouts would go on to form the bilateral dorsal longitudinalanastomotic vessels. By treating before this fusion and formationoccurs, the effect of each compound on the individual vessels as well asoverall patterning structural integrity (Isogai et al., Development2003, 130:5281) could be determined. Tg(mpo::EGFP) were tail fin clippedas previously detailed (Renshaw et al., 2006; Mahony et al., 2013;Beedie et al., Oncotarget 2016, 7(22):33237; Beedie et al., Mol. CancerTher. 2015, 14(10):2228) and incubated with test compounds or vehiclecontrol at 72 hours post fertilization. Fish were imaged at 24 hours andthe number of migratory neutrophils present at the wound site werecounted. Compounds inducing an at least 50% reduction of neutrophils tothe wound site were considered to have anti-inflammatory properties inthis system. All larvae were assessed for viability and morphologicalintegrity.

Chicken Embryology: Fertilized white leghorn chicken embryos wereincubated at 38° C. and staged according to Hamburger and Hamilton (HH)stages of development (1951). Embryos were tested at HH stage 17-18 (day2.5). Following membrane removal, test compounds or vehicle controlsolutions were applied globally over the embryo. The eggs were sealedand the development of the embryos was monitored up to HH stage 30 (E9).

Thalidomide Analogs: Compounds were dissolved in DMSO, and storedbetween 10-200 mg/mL, and used at a final working DMSO concentration of0.1%. The chemical structures of lead compounds of interest wereconfirmed by chemical characterization (purity >99.5%) and are shown inTables 2-5. The concentrations used to attain the data presented aregiven in Table 6; all compounds were screened across a range ofconcentrations from 1-200 μg/mL.

Imaging and Analysis: Imaging was performed using a Nikon MZ1500fluorescent stereomicroscope with a Nikon DS-5 digital camera, andanalyzed using Adobe Photoshop and Image J. Analysis was conducted usingPrism 6.0 (GraphPad Software, La Jolla, Calif.) and statisticalsignificance was assessed using two-tailed Student's t tests or ANOVAanalyses.

Example 1 Compound Synthesis

Exemplary syntheses are provided. Exemplary synthetic schemes are shownin FIGS. 1 and 2.

2-(2-Oxo-6-thioxo-3-piperidinyl)-4-nitro-1H-isoindole-1,3(2H)-dione (7)and 2-(2,6-Dithioxo-3-piperidinyl)-4-nitro-1H-isoindole-1,3(2H)-dione(9)

A mixture of 4-nitrothalidomide (412.0 mg, 1.359 mmol) and Lawessonreagent (1.21 g, 2.992 mmol) in toluene (200 mL) was stirred for 62hours under an atmosphere of nitrogen at reflux temperature. Afterremoving solvent, the residues were separated with chromatography onsilica gel (EA/PE=1/2) to afford product 7 (134.6 mg, 62.0%) and product9 (105.6 mg, 46.3%).

Compound 7 (yellow solid): mp 233.5-236.3° C.; ¹H NMR (DMSO-d₆) δ 12.66(s, 1H, NH), 8.33 (d, J=8.2 Hz, 1H, C5-H), 8.22 (d, J=7.8 Hz, 1H, C7-H),8.11 (t, J=8.2 Hz, 1H, C6-H), 5.32-5.27 (m, 1H, C3′-H), 3.24-3.12 (m,2H, C4′-H) and 2.50-1.96 (m, 2H, C5′-H) ppm; ¹³C NMR (DMSO-d₆) δ 210.9,167.2, 165.5, 162.8, 144.9, 137.3, 133.4, 129.3, 127.8, 123.0, 49.7,41.1 and 23.8 ppm; MS (CI/CH₄), m/z 319 (M⁺).

Compound 9 (red-brown solid): mp 200.5-202.0° C.; ¹H NMR (DMSO-d₆) δ13.82 (s, 1H, NH), 8.33 (d, J=7.8 Hz, 1H, C5-H), 8.23 (d, J=7.4 Hz, 1H,C7-H), 8.11 (t, J=7.8 Hz, 1H, C6-H), 5.37-5.32 (m, 1H, C3′-H), 3.35-2.80(m, 2H, C4′-H) and 2.70-2.12 (m, 2H, C5′-H); ¹³C NMR (DMSO-d₆) δ 206.6,201.2, 165.5, 162.9, 144.9, 137.3, 133.5, 129.3, 127.8, 123.0, 56.9,41.8 and 23.7 ppm; MS (CI/CH₄), m/z 335 (M⁺).

6-Isopropylamino-2-(3,4-dihydro-2-pyridone-3-yl)phthalimidine (46)

A mixture of 6-isopropylamino-2-(2,6-dioxo-3-piperidinyl)-phthalimidinehydrochloride (600 mg, 1.776 mmol) and lithium aluminum hydride (177.0mg, 4.664 mmol) in tetrahydrofuran (120 mL) was reacted for 80 minutesunder an atmosphere of nitrogen in an ice bath. Thereafter, hydrogenchloride in diethyl ether (1.0 M, 18 mL) was added at the sametemperature and was reacted for another 10 minutes. Acetic anhydride(2.7 mL) was added to the reactive system at 0° C., which was thengradually raised to room temperature and thereafter heated to reflux for20 minutes. After processing, the residues were isolated bychromatography on silica gel (CH₂Cl₂/MeOH=25/1) to afford product 46(46.1 mg, 18.2%) as a gum: ¹H NMR (DMSO-d₆) δ 9.40 (s, 1H, NH),7.70-7.35 (m, 3H, Ar—H), 6.05-5.95 (m, 1H, C6′-H), 5.12-5.03 (m, 1H,C5′-H), 4.95-4.75 (m, 2H, NH and C3′-H), 4.52 and 4.50 (AB system,J=17.8 Hz, 2H, C3-H) and 2.85-1.88 (m, 2H, C4′-H), 1.57 (s, 1H, Me₂CH)and 1.01-0.83 (m. 6H, CH₃CCH₃) ppm; ¹³C NMR (DMSO-d₆) δ 168.7, 167.7,142.3, 139.2, 134.2, 133.3, 126.0, 124.8, 109.5, 103.3, 50.8, 47.3,45.3, 25.0, 23.7 and 21.1 ppm; MS (CI/CH₄), m/z 285 (M⁺).

2-(6-Trifluoromethyl-benzothiazol-2-yl)-1H-isoindole-1,3(2H)-dione (59)

A mixture of phthalic anhydride (0.339 g, 2.289 mmol) and2-amino-6-trifluoromethyl benzothiazole (0.5 g, 2.291 mmol) in aceticacid (20 mL) was refluxed for 7 hours under an atmosphere of nitrogen.After removing solvent, the residues were recrystallized with acetone toafford product 59 (431.0 mg, 54.1%) as white needle crystals: mp279.0-281.5° C.; ¹H NMR (DMSO-d₆) δ 8.70 (s, 1H, C7′-H). 8.23 (d, 1H,C4′-H), 8.12-8.09 (m, 2H, C5,6-H), 8.02-7.99 (m, 2H, C4,7-H) and 7.88(d, 1H, C5′-H); ¹³C NMR (DMSO-d₆) δ 168.7, 160.0, 155.9, 140.1, 137.1,135.4, 130.0, 129.5, 128.7, 127.6, 127.3 and 124.5 ppm; MS (CI/CH₄), m/z348 (M⁺).

(1)-(2S,5R,6R)-3,3-Dimethyl-7-oxo-6-(1-oxo-1,3-dihydro-1H-isoindol-2-yl)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylicacid (65)

A mixture of phthaldialdehyde (1.34 g, 10.0 mmol) and(+)-6-aminopenicilanic acid (2.16 g, 10.0 mmol) in tetrahydrofuran (300mL) was stirred for 74 hours under an atmosphere of nitrogen at roomtemperature. After removing solvent, the residues were recrystallizedwith acetone to afford product 65 (416.0 mg, 12.5%) as a yellowishsolid: mp 197.5-199.5° C.; ¹H NMR (DMSO-d₆) δ 7.80-7.50 (m, 4H, Ar—H),5.93 (s, 1H, C6′-H), 5.68 (s, 1H, C5′-H), 4.91 and 4.68 (AB system,J=16.5 Hz, 2H, C3-H), 4.42 (s, 1H, C2′-H), 1.70 (s, 3H, CH₃) and 1.51(s, 3H, CH₃) ppm; ¹³C NMR (DMSO-d₆) δ 176.1, 173.2, 172.0, 146.7, 136.6,134.7, 132.6, 128.2, 127.5, 74.7, 71.4, 69.1, 65.1, 54.5, 35.0 and 31.1ppm; MS (CI/CH₄), m/z 334 (M⁺+2).

N-(4-Trifluoromethylpyridin-2-yl)-1-imino(4-trifluoromethylpyridin-2-yl)-isoindoline(67)

A mixture of phthaldialdehyde (41.4 mg, 0.308 mmol) and4-trifluoromethyl-2-aminopyridine (50 mg, 0.308 mmol) in tetrahydrofuran(10 mL) was stirred for 102 hours under an atmosphere of nitrogen atroom temperature. After removing solvent, the residues were separatedwith chromatography on silica gel (EA/Hex=1/3) to afford product 67(31.0 mg, 47.7%) as a gum: ¹H NMR (CDCl₃) δ 9.30-6.40 (m, 10H, Pht-H,Py-H) and 5.26 (s, 2H, C3-H) ppm; ¹³C NMR (CDCl₃) δ 162.4, 155.1, 153.3,150.1, 148.5, 141.0, 140.6, 139.9, 139.6, 131.8, 130.3, 127.7, 125.8,124.3, 123.8, 123.4, 114.1, 110.9 and 52.4 ppm; MS (CI/CH₄), m/z 422(M⁺).

2,3-Dihydro-2-(tricyclo[3.3.1.1^(3,7)]dec-2-yl)-1H-isoindol-1-one (72)

A mixture of phthaldialdehyde (134.1 mg, 1.0 mmol), 2-adamantylaminehydrochloride (187.7 mg, 1.0 mmol) and potassium carbonate (76 mg, 0.549mmol) in tetrahydrofuran (70 mL) was stirred for 8 days under anatmosphere of nitrogen at room temperature. After removing solvent, theresidues were purified with chromatography on silica gelMeOH/CH₂Cl₂=1/12) to afford product 72 (166.0 mg, 62.1%) as white needlecrystals: mp 121.5-123.0° C.; ¹H NMR (CDCl₃) δ 7.83 (d, J=7.7 Hz, 1H,C7-H), 7.54-7.41 (m, 3H, Ar—H), 4.67 (s, 2H, C3-H), 4.34 (s, 1H, C2′-H)and 2.49-1.71 (m, 14H, Ad-H) ppm; ¹³C NMR (CDCl₃) δ 169.3, 141.6, 133.0,131.0, 127.8, 123.3, 122.4, 58.3, 50.3, 38.2, 37.8, 32.7, 31.7, 27.6 and27.3 ppm; MS (CI/CH₄), m/z 267 (M⁺).

2,3-Dihydro-2-[1-(1-tricyclo[3.3.1.1^(3,7)]dec-1-yl)ethyl]-1H-isoindol-1-thione(74)

A mixture of2,3-dihydro-2-[1-(1-tricyclo[3.3.1.1^(3,7)]dec-1-yl)ethyl]-1H-isoindol-1-one(100 mg, 0.339 mmol) and Lawesson reagent (75.2 mg, 0.186 mmol) intoluene (18 mL) was stirred for 15.5 hours under an atmosphere ofnitrogen at reflux temperature. After removing solvent, the residueswere purified with chromatography on silica gel (EA/Hex=1/3) to affordproduct 74 (60.3 mg, 57.3%) as a yellow solid: mp 199.0-200.5° C.; ¹HNMR (DMSO-d₆) δ 7.85 (d, J=7.6 Hz, 1H, C7-H), 7.61-7.47 (m, 3H, Ar—H),5.06 (q, J=7.1 Hz, 1H, NCHMeAd), 4.86 (s, 2H, C3-H), 1.97-1.47 (m, 15H,Ad-H) and 1.21 (d, J=7.1 Hz, 3H, CH₃) ppm; ¹³C NMR (DMSO-d₆) δ 192.9,141.1, 139.4, 131.7, 128.4, 125.5, 123.0, 59.7, 55.5, 39.3, 37.7, 36.8,28.4 and 12.8 ppm; MS (CI/CH₄), m/z 309 (M-2); Anal. calcd for C₂₀H₂₅NS:C, 77.12; H, 8.09; N, 4.50. Found: C, 76.83; H, 8.03; N, 4.38.

2,3-Dihydro-4-nitro-2-(tricyclo[3.3.1.1^(3,7)]dec-1-yl)-1H-isoindol-1-one(76)

A mixture of methyl 2-methyl-3-nitrobenzoate (18.7 g, 95.8 mmol),N-bromosuccinimide (17.1 g, 96.1 mmol) and benzoyl peroxide (0.7 g, 2.9mmol) in carbon tetrachloride (500 mL) was refluxed for 20 hours. Thereaction mixture was cooled and concentrated. Thereafter, it wasprecipitated and washed with diethyl ether to afford methyl2-bromomethyl-3-nitrobenzoate (22.3 g, 84.8%) as yellowish crystals: mp68.0-70.0° C. (lit. 67.0-70.0° C.). A mixture of this compound (362.4mg, 1.322 mmol), 1-adamantylamine (200 mg, 1.322 mmol) and potassiumcarbonate (186.4 mg, 1.349 mmol) in DMA (2 mL) was stirred for 71 hoursunder an atmosphere of nitrogen at room temperature, and thencontinuously reacted for 23 hours at 50° C. After removing solvent, theresidues were purified with chromatography on silica gel (EA/Hex=1/3) toafford product 76 (224.8 mg, 54.4%) as a yellowish solid: mp214.0-215.5° C.; ¹H NMR (CDCl₃) δ 8.37 (d, J=7.6 Hz, 1H, C7-H), 8.12 (d,J=7.6 Hz, 1H, C5-H), 7.66 (t, J=7.6 Hz, 1H, C6-H), 4.93 (s, 2H, C3-H)and 2.43-1.73 (m, 15H, Ad-H) ppm; ¹³C NMR (CDCl₃) δ 166.1, 143.2, 138.0,136.5, 129.6, 129.4, 126.2, 56.2, 48.6, 40.0, 36.2, and 29.6 ppm; MS(CI/CH₄), m/z 312 (M⁺).

2,3-Dihydro-4-amino-2-(tricyclo[3.3.1.1^(3,7)]dec-1-yl)-1H-isoindol-1-one(77)

A mixture of compound 76 (100 mg, 0.320 mmol) and palladium on carbon(10 wt. %, 152.8 mg) in methanol (117 mL) was shaken for 48 hours underan atmosphere of hydrogen (45 lbs) at room temperature. After removingsolvent, the residue was purified with chromatography on silica gel(MeOH/CH₂Cl_(2=1/25)) to afford product 77 (88.8 mg, 98.2%) as ayellowish solid: mp 228.0-229.5° C.; ¹H NMR (DMSO-d₆) δ 7.08 (t, J=7.6Hz, 1H, C6-H), 6.75 (d, J=7.8 Hz, 1H, C7-H), 6.68 (d, J=7.7 Hz, 1H,C5-H), 5.31 (s, 2H, NH₂), 4.27 (s, 2H, C3-H) and 2.21-1.67 (m, 15H,Ad-H) ppm; ¹³C NMR (DMSO-d₆) δ 168.8, 143.7, 135.1, 128.9, 125.7, 115.9,110.1, 54.8, 46.0, 38.3, 36.4 and 29.5 ppm; MS (CI/CH₄), m/z 283 (M⁺+1).

2,3-Dihydro-2-(tricyclo[3.3.1.1^(3,7)]dec-1-hydroxy-3-yl)-1H-isoindol-1-one(86)

A mixture of phthaldialdehyde (134.1 mg, 1.0 mmol) and3-amino-1-adamantanol (167.3 mg, 1.0 mmol) in tetrahydrofuran (45 mL)was stirred for 7 days under an atmosphere of nitrogen at roomtemperature. After removing solvent, the residue was purified withchromatography on silica gel (MeOH/EA/PE=1/3/9) to afford product 86(115.0 mg, 40.6%) as white needle crystals: mp 218.5-219.5° C.; ¹H NMR(CDCl₃) δ 7.81-7.36 (m, 4H, Ar—H), 4.47 (s, 2H, C3-H) and 2.40-1.55 (m,15H, OH, Ad-H) ppm; ¹³C NMR (CDCl₃) δ 168.9, 140.8, 134.3, 131.0, 127.9,123.3, 122.4, 69.2, 57.8, 47.7, 44.1, 38.7, 34.9 and 30.8 ppm; MS(CI/CH₄), m/z 284 (M⁺+1).

Example 2 Effect of Thalidomide Analogs on In Vivo Angiogenesis inFli1:EGFP Zebrafish Embryos

To determine if the thalidomide analogs had any effect on angiogenesis,fli1:EGFP embryos (Lawson and Weinstein, Developmental Biology 2002,248:307-318) were exposed to either vehicle or a compound of interest,over a range of concentrations and over a range of time points startingat 24 hpf. Images were obtained at three time points over 24 hours afterexposure to the compounds. The best concentration and time point wereselected. CPS49, a compound that is chemically and structurally similarto thalidomide breakdown products, and is tetrafluorinated (which addsstability and bioactivity to the compound) and that causes severe limbdefects in chicken embryos, was used as a positive control. The embryoswere imaged at 48 hpf (FIGS. 3A3L) and analyzed for both vesselformation and the extent of growth of the vasculature. In comparison tocontrol zebrafish that showed normal patterning of the intersegmentalvessels, treated and responsive zebrafish showed a decrease in bloodvessel length and loss of vascular connectivity or a decrease in thenumber of forming blood vessels. The information acquired was collatedto define the overall impact upon angiogenesis and determine therelative anti-angiogenic activity of each compound (FIG. 4). In FIG. 4,the most highly anti-angiogenic compounds are in the lower left portionof the plot; the control (DMSO) lies in the midst of the scatter.Additionally the survival rates of the embryos were recorded as apercentage of the number of zebrafish alive following 24 hours ofexposure to each compound. (Table 6). The potency of each compoundvaried. For example, compound 4 was highly anti-angiogenic at 1.5 μg/mL,whereas compound 51 showed activity at 200 μg/mL (Beedie et al.,Oncotarget 2016, 7(22):33237). The results are based on the lowestconcentration of each compound producing an effect; compounds notproducing an effect are shown at the highest evaluated concentration(see Table 2 below).

Thirty-one compounds were anti-angiogenic as recorded by loss of ISVs orinhibition of ISV outgrowth in fli1:EGFP zebrafish. Of these compounds,18 were found to exhibit no anti-inflammatory properties in the mpo:GFPinflammatory assay and thus were classified as having anti-angiogeniconly properties in this in vivo assay (Table 2). The anti-angiogeniccompounds were found to inhibit vessel outgrowth and/or the number ofsprouting vessels at relatively low concentrations (1.5 μg/mL-200μg/mL), similar to ranges used in other screening studies (Tweedie etal., The Open Biochemistry Journal 2011, 5:37; Mahony et al., 2013;Beedie et al., manuscript in press). However, some anti-angiogeniccompounds were shown to induce death and defects within the treatedembryos (see Example 3). The analogs with potent anti-angiogenicactivity (for example, compound 4) also produced defects and highmortality rates in WT zebrafish and developing chicken embryo assays.This data complements other studies, which suggest the main cause ofthalidomide induced birth defects may be primarily via a loss of correctpatterning of the vasculature (Therapontos et al., PNAS 2009, 106:8573;Vargesson, Birth Defects Research Part C: Embryo Today: Reviews 2015,105(2):140; Beedie et al., Oncotarget 2016, 7(22):33237).

TABLE 2 Compounds having only anti-angiogenic activity Cpd Structure 4

11

14

17

19

34

51

55

60

69

70

73

81

83

84

89

91

93

Example 3 Effect of Thalidomide Analogs on Neutrophil Migration inResponse to Injury in Mpo:GFP Zebrafish

To assess the immunomodulatory effects of the thalidomide analogs, thempo:gfp transgenic zebrafish line (Renshaw et al., Blood 2006,108:3976-3978) was utilized because it is an accepted model ofinflammation, and can be used to test the effect of anti-inflammatoryagents. These embryos express GFP-tagged myeloperoxidase, where greenfluorescence marks the neutrophil cells involved in the inflammatoryresponse process, for example following injury, from 72 hours postfertilization (Renshaw et al., 2006; Mahony et al., 2013; Beedie et al.,Oncotarget 2016, 7(22):33237). At 72 hpf the dorsal third of the tailfin was removed and the embryos were immersed in the desired compound orvehicle (FIGS. 5A-5C). The embryos were examined for the effect on theinflammatory response quantified as an induction and migration ofneutrophils to the wound site 24 hours later. Control embryos with tailfin cuts showed a high neutrophil response (FIG. 5A). In comparison tocontrol embryos (FIG. 5A), compounds which did not exhibitanti-inflammatory properties (e.g., compound 51, 200 μg/mL) showed asimilar number of neutrophils in the wound site (FIG. 5B). Compoundswith anti-inflammatory properties (e.g., compound 20, 10 μg/mL; compound58, 10 μg/mL) showed at least a 50% reduction of neutrophils to thewound site (FIGS. 5C, 5D).

In total 21 compounds exhibited anti-inflammatory activity in this assay(FIG. 6), without anti-angiogenic effects (Table 3). Seven of thecompounds were found to be teratogenic (see Example 4). Compound 58 wasthe most effective analog at inhibiting the inflammatory response (FIG.6, Tables 3, 6). Compound 2(2,3-dihydro-2-(2-oxo-6-thioxo-3-piperidinyl)-3-thioxo-1H-isoindol-1-one)also possessed significant anti-inflammatory action (Beedie et al.,Oncotarget 2016, 7(22):33237).

TABLE 3 Compounds having only anti-inflammatory properties Cpd Structure2

 5*

7

9

24*

29*

32*

33*

46 

53 

56 

58 

59 

64 

65 

67*

72 

74 

76*

77 

86 

*Anti-inflammatory, but teratogenic in zebrafish and/or chicken embryoassay.

Thirteen compounds were anti-inflammatory and anti-angiogenic (Table 4).Given that tumors require a blood supply to survive, and that there arehigh levels of expression of cytokines in the tumor microenvironment(including Cyclooxygenase-2, TNF-α and interleukins), compoundstargeting both blood vessel formation and mediators of the inflammatoryresponse may be beneficial for anti-cancer therapy.

TABLE 4 Compounds with anti-angiogenic and anti-inflammatory activityCpd Structure 18

20

23

30

52

57

61

62

63

66

68

82

85

Example 4 Teratogenicity in Embryonic Development

To determine the effects of the candidate compounds on embryonicdevelopment, two in vivo model systems (developing zebrafish and chickenembryos) were utilized. The results indicated that of the potentialanti-inflammatory compounds, six had high mortality rates, and insurviving embryos treated with these compounds a teratogenic phenotypewas produced.

When the compounds with anti-angiogenic activity were screened in thesesystems, all of the embryos exhibited high mortality rates.Malformations in the development of the zebrafish embryo were noted,including inhibition of eye, otic vesicle and fin development (FIGS.7A-7F). In comparison to control wild type (WT) zebrafish (FIGS. 7A,7B), an anti-angiogenic compound (compound 4) induced defects (e.g.,microophthalmia and fin malformation) in the WT zebrafish (FIGS. 7C,7D), a compound (compound 8) with no effect in the assays (FIGS. 7E, 7F)and an anti-inflammatory compound (compound 77) had no apparent effecton the development of the zebrafish embryo (FIGS. 7G, 7H). Embryos werescreened at concentrations previously determined, in this study, to beanti-angiogenic or anti-inflammatory in the fli1:EGFP and mpo:GFP assaysrespectively.

Compounds of interest were screened in chicken embryos (FIGS. 7I-7P).FIG. 7I shows an embryo imaged in ovo with normal vasculature. Compound23 caused constriction in the vasculature of the chorioallantoicmembrane (white arrow) and necrosis (FIG. 7J). FIG. 7K shows a controlembryo imaged ex ovo. FIG. 7M shows a normal eye. Teratogenicity in thechicken embryo included microophthalmia (FIG. 7L—compound 80),hemorrhaging throughout the body and head (FIG. 7L, FIG. 7N—compound81), reduced body size (FIG. 7N), and limb and hand plate defects (FIG.7P). FIG. 7O shows a control forelimb. Compounds exhibitinganti-angiogenic properties produced more defects than those possessinganti-inflammatory only properties. In particular, chicken embryostreated with these compounds often showed signs of hemorrhaging (e.g.,compounds 80, 81) (FIGS. 7L, 7N) and constriction of the vasculature andnecrosis in the surrounding chorioallantoic membrane (e.g., compound 23)(FIG. 7J). An embryo treated with compound 11 had missing digits in thehindlimb.

All of the anti-angiogenic compounds of Table 2 were shown to beteratogenic in the developing zebrafish and chick model systems. All ofthe compounds exhibiting both anti-angiogenic and anti-inflammatoryproperties (Table 4) also were shown to the teratogenic in the WTzebrafish and developing chick model systems.

Table 5 provides structures of compounds used in the foregoing examples.Table 6 provides concentrations and n numbers used in the foregoingexamples that exhibited activity. Compounds exhibiting anti-angiogenicproperties are shown at the lowest concentration producing a response.Compounds that did not produce a response in this assay are shown at thehighest concentration tested, or the highest concentration where thesurvival rate was not affected. Compounds which significantly inhibitedneutrophil migration to the wound site are shown at the lowestconcentration with activity. Compounds that did not produce a responsein this assay are shown at the highest concentration tested, or thehighest concentration where the survival rate was not affected.Concentrations used in the WT zebrafish and chicken embryo assays toassess for teratogenesis, were equal to or higher than those utilized inthe fil1:EGFP and mpo:GFP zebrafish assays

TABLE 5

2 C₁₃H₁₀N₂O₂S₂ MW 290.36

4 C₁₃H₁₀N₂OS₃ MW 306.43

5 C₁₃H₉N₃O₆ MW 303.23

7 C₁₃H₉N₃O₅S MW 319.29

9 C₁₃H₉N₃O₄S₂ MW 335.36

11 C₁₃H₁₁N₃O₄ MW 273.24

14 C₁₃H₁₀N₂O₄S MW 290.29

17 C₁₃H₁₀N₂O₄S MW 290.29

18 C₁₅H₁₂N₂O₆ MW 316.27

19 C₁₅H₁₂N₂O₅S MW 332.33

20 C₂₀H₁₆N₂O₅ MW 364.35

23 C₁₃H₉ClN₂O₃S MW 308.74

24 C₁₃H₁₁N₃O₅ MW 289.24

29 C₁₃H₁₃N₃O₂S MW 275.33

30 C₁₃H₁₃N₃O₂S MW 275.33

32 C₁₃H₁₃N₃OS₂ MW 291.39

33 C₁₃H₁₁N₃O₅ MW 289.24

34 C₁₃H₁₃N₃O₃ MW 259.26

45 C₁₆H₁₉N₃O₂ MW 285.34

46 C₁₆H₁₉N₃O₂ MW 285.34

48 C₁₉H₁₉N₃O₅ MW 369.37

51 C₂₂H₁₇N₃O₃S₂ MW 435.52

52 C₂₁H₂₅N₃O₃S₂ MW 431.57

53 C₂₀H₂₃N₃O₃S₂ MW 417.54

55 C₁₃H₁₂N₄O₂ MW 256.26

56 C₁₃H₁₁N₃O₃ MW 257.24

57 C₁₂H₉N₃O₄ MW 259.22

58 C₁₂H₉N₃O₄ MW 259.22

59 C₁₆H₇F₃N₂O₂S MW 348.30

60 C₁₆H₆F₃N₃O₄S MW 393.30

61 C₁₆H₇F₃N₂O₃S MW 364.30

62 C₁₆H₉F₃N₂OS MW 334.32

63 C₁₆H₈F₃N₃O₃S MW 379.31

64 C₁₆H₁₄N₂O₅S MW 346.36

65 C₁₆H₁₆N₂O₄S MW 332.37

66 C₂₀H₁₂F₆N₄ MW 422.33

67 C₂₀H₁₂F₆N₄ MW 422.33

68 C₁₅H₁₀F₃NO MW 277.24

69 C₁₈H₂₁NO MW 267.37

70 C₁₉H₂₃NO MW 281.39

71 C₂₀H₂₅NO MW 295.42

72 C₁₈H₂₁NO MW 267.37

73 C₁₇H₁₉NO MW 253.34

74 C₂₀H₂₅NS MW 311.48

76 C₁₈H₂₀N₂O₃ MW 312.36

77 C₁₈H₂₂N₂O MW 282.38

81 C₂₀H₂₆N₂O MW 310.43

82 C₁₈H₂₀N₂O₃ MW 312.36

83 C₁₈H₂₂N₂O MW 282.38

84 C₁₇H₁₈N₂O₃ MW 298.34

85 C₁₇H₂₀N₂O MW 268.35

86 C₁₈H₂₁NO₂ MW 283.36

89 C₂₀H₂₄N₂O₂S MW 356.48

90 C₁₈H₂₂N₂O₃ MW 314.38

91 C₁₈H₂₄N₂O MW 284.40

93 C₁₈H₂₄N₂O MW 284.40

TABLE 6 The results shown are based on the lowest concentration of eachcompound producing an effect; compounds not producing an effect areshown at the highest evaluated concentration. Assay fli1:EGFP mpo:GFPangiogenesis inflammation WT n n Zebrafish Chicken Com- (μg/ (survived/(μg/ (survived/ (μg/ (μg/ pound mL) tested) mL) tested) mL) n mL) n 41.5  4/11 10 19/23 10 15 100 10 11 50 24/43 10 16/22 10 15 100 6 14 10 7/23 10 14/24 10 15 100 6 17 15 26/50 10  5/5  10 4 100 3 19 1 13/51 1 5/12 1 18 50 3 34 100 13/20 100  7/9  100 20 100 5 51 200  7/25 1  4/11100 3 55 100  6/10 200  6/11 100 3 60 10  3/10 1  5/15 100 3 69 10  6/14200  8/17 100 3 70 10  8/10 200 10/15 100 3 73 10  6/12 10 7/13 100 3 8150  7/8  50  9/21 10 7 100 12 83 20  3/12 20  6/23 10 15 100 4 84 50 2/11 10 12/21 10 4 100 4 89 10  7/11 100 3 91 10  5/18 100 3 93 10 8/10 100 3 2 10 21/24 10 26/30 10 11 100 12 7 10 28/33 10 29/29 10 15100 3 9 10 14/21 10 12/12 10 5 100 3 46 10 14/30 10 24/50 100 3 53 10 9/14 10 13/19 100 7 100 3 56 10  9/19 10  7/8  50 8 58 10 33/33 5  7/16100 3 59 10 21/32 100  6/11 100 5 50 6 64 10  8/15 10  8/15 100 3 65 20013/13 200 10/20 100 3 72 200  9/20 10  6/15 100 3 74 10  9/15 200  6/14100 3 77 10 43/62 200  5/21 100 39 100 3 86 200  4/12 100 14/19 100 3 57.5  8/14 10 28/28 15 15 100 3 24 50 13/25 50  6/6  50 5 100 3 29 10 6/17 5  2/10 50 11 32 100 21/25 10  6/15 50 15 100 3 33 100 15/21 10 4/15 50 15 100 5 67 10 13/22 200  6/15 100 3 76 10 28/32 100  5/11 1003 18 50 24/43 50 14/14 50 15 100 6 20 10 51/79 10  8/14 10 18 50 6 23 1023/30 10 13/35 10 15 100 3 30 10  4/8  10 10/10 10 27 100 3 52 100  6/7 100  7/21 100 3 57 8.5  8/12 17  8/15 100 3 61 100  3/15 1  9/20 100 362 10 18/20 10  6/20 100 3 63 200  6/23 100 12/20 50 3 66 100 15/15 100 6/15 100 3 68 10 22/34 100  5/15 50 4 82 5 18/27 5  8/22 5 8 100 8 8510  7/20 100 11/20 100 3

Example 5 Cellular Proliferation, Nitrite, and TNF-α Protein LevelQuantification

The CellTiter 96® AQueous One Solution Cell Proliferation Assay(Promega, Madison, Wis.) is routinely used as an assay of cellproliferation, and was used according to the manufacturer'srecommendations. Changes in cellular health status are determined by useof indirect measures related to the formation of a colored tetrazoliumdye product that can be measured spectrophotometrically at 490 nm λ. Anelevation in absorbance is indicative of an increase in cell number and,hence, cellular proliferation. Optical densities (expressed as O.D.s)were measured after various incubations.

Nitrite levels in the culture media were measured by use of the GriessReagent System (Promega, Madison, Wis.), following the manufacturer'sprotocol. The O.D. of unknown samples was read at 520 nm λ, compared toa sodium nitrite standard curve (1.5 μM to 100 μM) and nitrite measuredmedia concentrations expressed as μM units. As the lowest nitriteconcentration on the Griess Reagent System standard curve was 1.5 μM,this was chosen as the effective cutoff for defining measurable nitriteconcentrations. TNF-α protein levels were measured by use of an ELISAspecific for mouse TNF-α protein (BioLegend, San Diego, Calif.) and areexpressed as a % change from their appropriate control or as pg/ml.

RAW 264.7 cells obtained from ATCC (Manassas, Va., USA) were grown inDMEM media supplemented with 10% FCS, penicillin 100 μg/ml andstreptomycin 100 μg/ml, and maintained at 37° C. and 5% CO₂. Cells wereseeded in 24 well plates and, 24 hours later, were utilized in studies.One hour prior to the initiation of any study, the seeding media wasreplaced with fresh media (1 mL), and the cells were allowed toequilibrate at 37° C. and 5% CO₂.

RAW 264.7 cells were challenged with LPS (Sigma, St Louis, Mo.: serotype055:B5) from at a final concentration of 30 or 60 ng/mL. Thisconcentration of LPS in RAW 264.7 cells induces a sub-maximal rise inboth TNF-alpha and nitrite levels without a loss in cellular viability.This sub-maximal rise is useful for assessing whether the addition of anexperimental drug can either lower or further raise levels of TNF-α andnitrite. Twenty four hours following the addition of LPS, conditionedmedia was harvested and analyzed for quantification of secreted TNF-αprotein and nitrite levels. Fresh media was replaced into the wells andcell viability was then assessed.

Thalidomide and analogs were prepared in tissue culture grade DMSO(Sigma). RAW 264.7 cells were pretreated with thalidomide, analogues orvehicle one hour prior to a challenge with LPS. The effects of variousconcentrations of thalidomide analogues were assessed. All compoundswere synthesized to a chemical purity of >99.5%, as assessed by chemicalcharacterization by a combination of 1H NMR, 13C NMR and GC/MS analyses(Bruker AC-300 spectrometer, together with elemental analyses (AtlanticMicrolab, Inc., Norcross, Ga.).

Data throughout are expressed as means±standard errors, where the nnumber is shown in parentheses. The n number refers to the number ofwells in the tissue culture plates. Statistical comparisons wereundertaken by use of either a Students t-test, or by One Way ANOVA withappropriate Bonferroni corrections for multiple comparisons, as required(GraphPad InStat Version 3.05). P values of <0.05 are considered to beof statistical significance, *, **, *** refer to P<0.05, P<0.01 andP<0.001 respectively. The results of representative compounds 7, 9, 59,62, 64, 65, 68, 72, 74, 77, 81, 86, and 98 are shown graphically inFIGS. 8A and 8B. Additional results are shown in Tables 7-12; NS=notsignificant; a “+” sign=anti-inflammatory or increased cell numbers; a“−” sign=cell toxicity or pro-inflammatory.

TABLE 7 1 μM 10 μM 30 μM Drug # TNF-α Nitrite Viability TNF-α NitriteViability TNF-α Nitrite Viability 5 NS NS NS NS NS NS NS NS NS 6 NS NSNS NS NS NS NS − NS 7 NS NS NS NS NS NS NS + NS 8 NS NS NS 9 NS + NS 60μM 100 μM TNF-α Nitrite Viability TNF-α Nitrite Viability 5 NS NS NS NSNS NS 6 NS NS NS NS + 7 NS + NS + + + 8 NS NS NS NS NS NS 9 NS + NS NS ++

TABLE 8 Drug 3 μM 10 μM 30 μM # TNF-α Viability TNF-α Viability TNF-αViability 28 NS − NS − + − 29 NS − NS − NS − 31 NS NS + NS + NS 32 +NS + NS + NS 35 NS NS NS NS NS NS 36 NS NS NS NS + NS 37 NS NS NS NS +NS 38 NS NS NS NS NS NS 39 NS NS NS NS NS NS 40 NS NS NS NS + NS 41 +NS + NS + NS 43 NS NS NS NS NS NS 44 NS NS NS NS + NS 54 NS NS + NS + −106 NS + NS + NS +

TABLE 9 Drug 1 μM 10 μM 30 μM # TNF-α Viability TNF-α Viability TNF-αViability 47 + NS + NS + NS 48 + NS + NS + NS 49 + NS NS NS + NS 50 NS− + NS NS NS 51 + NS + NS + − 52 + NS + NS + NS 53 + − + NS NS +

TABLE 10 1 μM 10 μM 30 μM Drug # TNF-α Nitrite Viability TNF-α NitriteViability TNF-α Nitrite Viability 57 NS NS NS NS + NS NS + NS 58 NS + NSNS + NS NS + NS 59 NS + NS NS NS NS + + NS 61 NS NS NS NS + NS + + − 62NS NS NS NS NS NS NS + NS 63 NS − NS NS − NS NS − − 64 NS NS NS NS NS NSNS NS NS 65 − NS NS − NS NS − NS NS 66 NS NS − NS NS NS + + − 67 NS NSNS NS NS NS NS + NS 68 NS NS NS NS NS NS NS NS NS 69 NS NS NS NS NS NSNS NS NS 70 NS NS NS + NS NS + + NS 71 NS NS NS NS NS NS + + − 72 + NS +NS NS NS + + − 73 NS NS NS NS + NS + + − 74 NS NS NS NS NS NS NS + NS 75NS NS NS + NS NS + NS NS 76 NS NS NS NS + NS + + NS 77 NS NS NS NS NS NSNS NS NS 78 NS NS NS + NS NS + + − 79 NS NS NS NS NS NS NS + − 80 NS NSNS + + − + + NS 81 NS NS NS NS NS NS + + NS 82 NS + NS + + − + + − 83 NSNS NS NS + NS NS + NS 84 NS NS NS NS + NS NS + NS 85 NS NS NS NS NS NSNS − NS 86 NS NS NS + NS NS + NS NS 87 NS NS NS NS NS NS NS NS NS 88 NSNS NS NS NS NS NS NS NS 89 NS NS NS NS + NS NS + −

TABLE 11 1 μM 10 μM 30 μM Drug # TNF-α Nitrite Viability TNF-α NitriteViability TNF-α Nitrite Viability 90 + + NS + + NS + + NS 91 NS +NS + + + + + + 92 NS + NS + + − + + − 93 NS NS NS NS NS NS NS + NS 94NS + NS + + −− + + − 95 NS NS NS NS NS + NS + + 96 NS + − + + − + + NS97 NS NS NS + NS NS NS + NS 98 SIG + NS + + NS + + NS 99 NS + NS NS +− + + − 100 NS NS NS NS NS NS NS NS NS 101 NS + − NS + − + + NS 102 NSNS NS NS NS NS NS NS NS 103 NS + NS + + NS SIG NS SIG 104 NS − NS NS −NS NS − NS 105 NS NS NS NS + NS + + −

TABLE 12 10 μM 30 μM 60 μM Drug # TNF-α Nitrite Viability TNF-α NitriteViability TNF-α Nitrite Viability  111 NS NS NS NS NS NS NS − NS  112 NSNS − + + − + + −  113 NS NS − + + − + + −  114 + + − + + − + + − 115 + + − + + − + + −  116 NS NS − NS NS − NS NS NS  117 + + − + +− + + −  118 + + − + + − + + −  119 + + − + + − + + −  120 + + − + +− + + − *121 NS + NS + + NS *Drug 121 at 100 μM: TNF-α = +, Nitrite = +,Viability = NS

Example 6 Rat Aortic Ring Assay of Angiogenesis

Matrigel® matrix (BD Biosciences) was defrosted overnight on ice. Thewells of a 24 well plate were coated with 300 μL of the gel using frozenpipette tips. The gel was left to set at room temperature. Whenrequired, six week old male Sprague Dawley rats were euthanized by CO₂and decapitation. The blood was drained from the tissue and thedescending aorta dissected. The aorta was cleaned in EBM media, the overlying fascia removed, and the tissue was sliced into 1-mm sections usinga sterile scalpel and tweezers. The sections were rinsed in clean EBMthen placed onto the coagulated gel. Another 300 μL of Matrigel® matrixwas pipetted onto the ring and left to set for 30 minutes. EGM™-IIendothelial growth medium (500 μL) was added to each well. The ringswere incubated overnight at 37° C. The next day the media was removedand replaced with EBM containing compounds (50 μM) or media. TNP-470((Chloracetyl) carbamic acid(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2.5]oct-6-ylester) was used as a positive control. The rings were incubated in thecompounds for 5 days. On day 5 the rings were imaged using an EVOS™scope. The vessel outgrowth was quantified by binary conversion in ImageJ open-source image processing software and average outgrowth calculated(FIG. 9). Compound 86 reduced outgrowth the most in this assay (17.3%outgrowth).

Example 7 HUVEC Lattice Formation

ECMatrix™ gel solution and 10× diluent buffer (Millipore) were defrostedslowly overnight on ice. One part diluent was added to nine parts gelusing frozen pipette tips. 100 μL of the ECMatrix™ solution was slowlyadded to the required wells in a 96 well plate and left to set for onehour. HUVEC cells (human umbilical vein endothelial cells) wereharvested and counted. The cells were resuspended and plated at 100000cells/well in 100 μL of RPMI medium with thalidomide analogs (1-30 uM)or DMSO. The plates were returned to the incubator and analysed after 18hours of exposure. The extent of inhibition of lattice formation wasassessed using ImageJ Results are shown for 1 μM, 10 μM, and 30 μMconcentrations of the thalidomide analogs (FIGS. 10A and 10B).

Example 8 Anti-Cancer Screening

As well as being immunomodulatory and anti-angiogenic, lenalidomide andpomalidomide are directly cytotoxic to cancer cells. To establish if thelead compounds exhibited similar abilities, Compounds 29 and 86underwent screening in 59 cancer cell lines. Compounds 29 and 86 werescreened at a concentration of 1×10⁻⁵ M (FIGS. 11 and 12, respectively).Calculated values from 0-100 indicate growth inhibition relative to ano-drug control, and values less than 0 indicate lethality, relative tothe number of cells at time zero. A value of 100 means no growthinhibition. A value of 40 means 60% growth inhibition. A value of 0means no net growth over the course of the experiment. A value of −40means 40% lethality. A value of −100 means all cells are dead. With theexception of leukemic cell lines, both compounds exhibited very littleactivity in this assay. The lack of anti-cancer activity for compounds29 (FIG. 11) and 86 (FIG. 12) suggests they are targeting thevasculature directly, and are not simply cytotoxic to the cells.

Example 9 Anti-Angiogenic Activity in an Ex Vivo Model

The saphenous vein was cleaned of subcutaneous tissue and embedded inMatrigel® matrix as previously described. After 10 days of incubation,the transverse sections of the veins were imaged. The outgrowth betweenthe vehicle and no treatment (media) control was equivalent(106.65%±20.14, n=2 and 103.35%±4.06, n=2 respectively; FIGS. 13A and13B). Treatment with TNP-470 caused a vast reduction in vessel outgrowth(9.44%±1.65, n=3; FIGS. 13A and 13C (vein section plated on its side).When the thalidomide analogs 29 and 86 were tested, there was a cleardistinction in the activities of the compounds. Compound 86 was able toinhibit outgrowth at all concentrations (FIGS. 13A and 13E (10 μM); n=3per concentration). At 5 μM (155.12%±23.69, n=3) 10 μM (168.06%±14.87,n=2) and 50 μM (102.38%±16.78, n=3), there was no significant decreasein vessel outgrowth by compound 29 (FIGS. 13A and 13D (10 μM)). At highconcentrations (100 μM) compound 29 had a limited anti-angiogenic effect(62.53%±13.69, n=3; FIG. 13A).

Example 10 Maximum Tolerated Dose and In Vivo Efficacy

Compounds 29 and 86 were selected for in vivo efficacy studies of humanprostate cancer. Mice positive for the severe combined immune deficieny(SCID) spontaneous mutation Prkdc^(scid) are deficient in functional Tcells and B cells, have reduced levels of gamma globulins andlymphopenia (low white blood cell count). Due to their comprised immunesystem, SCID mice are amenable to xenogeneic grafts. Before starting theefficacy study, a maximum tolerated dose was established for eachcompound. Compound administration was by tail vein injection (IV) with a27-gauge needle. A volume of 100 uL per 25 g of body weight was injectedfor each compound and dose. Compounds 29 and 86 were formulated in 12%DMSO and 88% TPGS (tocopherol polyethylene glycol succinate) indistilled sterile water. This formulation was well tolerated in vehiclecontrol mice. The maximum tolerated dose was defined as the highest doseat which 100% of animals could tolerate treatment with mild or notoxicity for a minimum of one week of treatment. Initially, eachcompound was administered 3× per week to five mice per group, at 5 and10 mg/kg. Each dose level was monitored for at least one week. At 20mg/kg, the compounds were seen to precipitate in the solution. Animalswere weighed daily during the treatment courses. At 5 mg/kg and 10mg/kg, no animals died or needed to be euthanized due to drug-relatedtoxicity and no significant weight loss was observed for any treatmentgroup (FIG. 14). The selected dose for further experimentation wastherefore 10 mg/kg.

When the tumors of SCID mice xenografted with PC3 cells were palpable,mice were dosed with 10 mg/kg of compound 29 or compound 86, or giventhe vehicle alone. From day 0 to day 24, the tumor sizes invehicle-treated animals increased by 301.33% (FIG. 15, tumorsize=401.33%±35.98%). No reduction in tumor burden was observed foreither treatment group. However, there was a trend to separate betweenthe vehicle treated and compound 86 treated mice (p=0.07), suggesting aninhibition of tumor growth rather than reduction of tumor burden.

Example 10 Models for Screening and/or Evaluation of Thalidomide Analogsfor Treatment of Alzheimer's Disease, Traumatic Brain Injury, IschemicStroke, Multiple Sclerosis, and Systemic Inflammatory Disorders

Evaluation of potential actions in Alzheimer's disease (AD) can beappraised in transgenic mouse models of AD that include APP+PS1 genemutations that result in the excessive production of the AD toxicpeptide amyloid-β (Aβ), as described within Tweedie et al. (JNeuroinflamm 9:106, 2012; Gabbita J Neuroinflamm. 9:99, 2012). For suchmice in a time-dependent manner, Aβ is generated and aggregates in brainas amyloid plaques that can be appropriately quantified biochemicallyand/or stained for visualization by immunohistochemical techniques (seeTweedie et al., J Neuroinflamm 9:106, 2012). Elevations inphosphorylated-tau and reductions in synaptic markers (for example,SNAP25 and synaptophysin) are additionally evident, as are elevations inmarkers of neuroinflammation (for example, CD68-positive microglia) thatassociate with impairments in measures of cognitive performance (as forexample measured by the Morris water maze). The daily administrationover multiple weeks (for example, 6 weeks) of a well-tolerated compoundthat lowers TNF-α can result in mitigation of many of these aberrantchanges, as compared to untreated mice and/or younger mice of the samebackground.

Evaluation of actions of anti-inflammatory agents and in particularthose that lower TNF-α generation can be valuably appraised across anumber of animal models of mild and moderate traumatic brain injury(TBI), which represent the majority of cases that present in humans. Theweight drop model, one of the original animal models of TBI, involvesdropping a free-falling weight guided by metal tubing to impact thetemporal brain region of an anesthetized animal (for mice such a weightis often 30 or 50 g from approximately 80 cm (31.5 inches) height, whichrepresents concussing an animal with approximately its own weight inmuch the same manner as the banging of heads in human contact sports ora fall in the elderly. Anesthesia for this weight drop model is bestachieved by use of a short-acting gaseous anesthetic (for example,isoflurane) delivered immediately before the injury, contributing tothis method's ease of preparation. This range of weight generates aninjury that is free from gross neuroanatomical changes (Tweedie et al.,J Neurosci Res. 85:805-15, 2007) and skull fracture complications thatcan occur with heavier weights. For this model, the anesthetized mouseis placed below the guide tube through which the weight is dropped,aligned so that it will strike the skull on the temporal side, betweenthe corner of the eye and the ear (predefined on either the left orright side). The head rests on a supportive foam sponge positioned toallow antero-posterior motion without any rotational head movement atthe moment of impact (Rachmany et al., PLoS ONE, 8:e79837, 2013; Baratzet al., J Neuroinflamm 12:45, 2015). The resulting pathology of theweight drop technique is neurodegeneration with diffuse neuronal celldeath and neuroinflammation throughout cortical areas and hippocampus ofthe impacted side of the brain. This can be evaluated by biochemicalassays of TNF-α or other proinflammatory cytokines, and byimmunohistochemical analysis of markers of degeneration elevated.Fluoroiade B or C staining) with and without neuronal marker (e.g.,NeuN) quantification, and by quantification of astrocyte/microgliaactivation (e.g., elevated glial fibrillary acidic protein and/or Iba1antibody staining). Chief symptoms of mild TBI in humans as well as theweight drop rodent model of the disorder include cognitive impairments,particularly in measures of spatial and visual memory that can bequantified in rodents with behavioral tests (specifically by employingnovel object recognition and Y-maze paradigms, respectively (Deselms etal., J Neurosci Methods 2016). The administration of an effective amountof a TNF-α lowering drug, optionally in conjunction with ananti-inflammatory drug, can mitigate behavioral impairments measured asearly as within 7 days or after longer durations (i.e., 30 days) and canmitigate elevations in proinflammatory cytokine levels, neuronalapopotosis and markers of neuroinflammation, as utilizing paradigms usedwithin Baratz et al., (J Neuroinflamm 12:45, 2015). In rodents, theexperimental drug is administered by any suitable route, e.g.,systemically, as a single dose or multiple doses within 12 hr ofinduction of brain injury. The timeframe for administering theexperimental drug may be different in species other than rodents; forexample, the timeframe may be substantially longer for administration tohumans.

For moderate TBI, rodent models including controlled cortical impact(CCI) and fluid percussion injury (FPI) models can be used to evaluateefficacy of anti-inflammatory/TNF-α lowering agents, as detailed withinWang et al., (J Neuroinflamm 13(1):168, 2016) and Eakin et al., (PLoSOne. 8(12):e82016, 2013). In these well-characterized animal models ofbrain injury, a mechanical injury force is directly applied to thesurface of the brain, and thus a craniectomy is required before injuryinduction in either the anesthetized mouse or rat (Deselms et al., JNeurosci Method 2016). For CCI this is delivered by a rigid impactorusing an instrument that is precisely adjustable over the parameters oftime, velocity and depth (Wang et al., J Neuroinflamm 13(1):168, 2016),whereas for FPI a fluid-mediated pressure pulse is generated by aclassical piece of equipment. This comprises a cylindrical reservoirthat is filled with sterile isotonic saline, which on one end has atransducer connected via a tube and male Luer lock to a female Luer lockattached to the skull. Injury is initiated by the development of anacute fluid pulse against the intact dural surface, which is induced bythe release of a pendulum (from a precalibrated height or angleassociated with a desired injury severity) that strikes a piston at theother end of the reservoir (Eakin et al., (PLoS One. 8(12):e82016, 2013;Deselms et al., J Neurosci Method 2016). Both models can induce gradedlevels of brain injury and create axonal injuries and contusions,accompanied by impaired neurologic motor function and cognitiveimpairments. This is accompanied by immediate necrotic cell death and,more importantly, by a slower apoptotic cell death that is amenable toreversal within cortical and hippocampal regions (Yang et al., ExpNeurol. 269:56-66, 2015; Yang et al., Neurobiol Dis 2016). Appraisal ofefficacious anti-inflammatory/TNF-α lowering agents is evaluated bytheir administration (e.g., systemically, whether by intravenous oranother route) at a time up to 5 to 7 hr following injury in rodents.The timeframe may vary in other species; for example, the treatmentwindow may be longer in humans. Evaluation of brain inflammation andneuronal loss is made by biochemical and immunohistochemical assays, asdetailed in Wang et al., (Neuroinflamm 13(1):168, 2016), and behavioralmeasures include functional outcomes as revealed by (i) motor asymmetrymeasured by elevated body swing test, (ii) motor coordination andbalance assessed by a beam walking test, (iii) a neurological severityscores (mNSS) to provide a composite evaluation of overall neurologicfunction, and (iv) analysis of sensory/motor function by quantifying thelatency for animals to remove an adhesive sticker from their forepaw—contralateral to the side of brain injury (as detailed in Yang etal., Exp Neurol. 269:56-66, 2015; Yang et al., Neurobiol Dis 2016).Cognitive assessment likewise can be made that show impairments, asevaluated by Morris Water Maze (as detailed in Eakin et al., PLoS One.8(12):e82016, 2013), which is amenable to rescue by an effectivetherapy.

A further model to evaluate efficacy and functional outcome ofanti-inflammatory/TNF-α lowering agents following brain damage can beundertaken in the classical rodent model of focal ischemic strokeinduced by middle cerebral artery occlusion/reperfusion (MCAO/R)performed via the intraluminal suture technique in an anesthetized mouseor rat with 1 hr of MCAO, as described by Yoon et al., (J Neurosci Res.91(5):671-80, 2013). In this animal model of ischemic stroke,experimental therapeutics are evaluated after administration by anysuitable route, such as by systemically, at times either prior to orfollowing MCAO/R (for example up to 3 hr after injury).Immunohistochemical and biochemical analyses then undertaken at 24 to 72hr to evaluate markers in neuroinflammation (for example, GFAP and Iba1staining, and/or evaluation of TNF-α brain levels), area/volume ofinfarction (2,3,5-triphenyltetrazolium chloride (TTC) staining).Additionally, behavioral studies can reveal efficacious drug effects(for example by evaluation of a 5-point neurological deficit score (0,no deficit; 1, failure to extend left paw; 2, circling to the left; 3,falling to the left; 4, unable to the walk spontaneously) assessed in ablinded manner).

In a further example, efficacy of compounds can be evaluated in ananimal model of multiple sclerosis involving experimental autoimmuneencephalomyelitis (EAE), as described in Eitan et al., (Exp Neurol.273:151-60, 2015). EAE is induced with MOG 35-55 peptide (HookeLaboratories, Inc.; kit EK-2110), whereby on day 0, mice receive twosubcutaneous injections of MOG 35-55 mixed with an adjuvant and 2 hlater pertussis toxin is injected intraperitoneally. One day later(i.e., experimental day 1) a second pertussis toxin injection isadministered, and then mice are evaluated daily over an extended time(for example, 23 days) with the provision of readily available soft foodand water in the cages when severe motor impairment occurs. In additionto following weight, behavioral scoring can be time-dependentlyundertaken to evaluate efficacious experimental drug treatment effects,and classical biochemistry and immunohistochemistry can be undertaken(to measure, for example, cytokine levels, markers of neuroinflammation(GFAP, Iba1), myelination (as for example by myelin basic proteinantibody levels). As an example of a behavioral assessment of value todefine efficacious drug actions, a widely-employed six-point scale canbe used as follows: 0) no evidence of motor impairment, tail is erect,and mouse spreads hind legs when picked up by the tail; 1) tail is limpwhen the mouse is picked up by the base of the tail; 2) limp tail,weakness in hind legs/wobbly gait, and legs closed when picked up by thetail; 3) limp tail with paralysis of one front and one hind limb; 4)limp tail, complete hind limb paralysis, and partial forelimb paralysis;and 5) mouse is found dead or must be euthanized due to severeparalysis. Additionally, motor evaluations can be made by use of a rotorrod, as detailed in Eitan et al., (Exp Neurol. 273:151-60, 2015).

Systemic disorders that have an inflammatory involvement additionallycan be evaluated for efficacious drug actions, as for example in animalmodels of sarcopenia involving hind limb suspension to induce musclewasting. To achieve muscle unloading by hindlimb suspension, mice can beanesthetized (i.e., 2.5% isoflurane) and their tails cleaned withrubbing alcohol and air dried, covered with a light coat of benzointincture, and dried with a hair dryer until tacky. Strips of elastoplastadhesive bandage can then be applied to the proximal two-thirds of allsides of the tail and looped through a swivel attachment mounted abovethe cage designed to allow the animal to move rotationally 360° withonly the forelimbs able to come into contact with the cage floor. Theanimals are provided food and water ad libitum and monitored daily forsigns of lethargy or illness. Evaluation of efficacy of experimentaldrugs is made following their administration daily, e.g., systemicadministration, over an extended period of time with efficacy measuresrelated to leg skeletal muscle weight, as well as biochemical,immunohistochemical and functional measures. As little as 5 days ofmuscle unloading can induce atrophy (Fix et al., J Appl Physiol 2016).Biochemical and immuno-histochemical analyses can be made of musclestructure (including Western blot analysis of key muscle proteins), aswell as quantification of capillary networks and mitochondria number andfunction. In addition, functional evaluations (such as defined measuresof gait) can be quantified. These evaluations can be made during muscleunloading and, in particular, during reloading of muscle to evaluate thenormalization of muscle structure following its wasting. Notably, TNF-αis chronically elevated in sarcopenia, and its higher levels arestrongly correlated with muscle loss, reduced strength and morbidity(Schaap et al., J Gerontol-Series A Biol Sci & Med Sci 64: 1183-9, 2009;Fan et al., Mediators Inflamm. 2016:1438686, 2016). TNF-α inducesskeletal muscle loss through increased myofibrillar protein degradationand cell apoptosis, and thus induces muscle atrophy and the inhibitionof muscle regeneration following injury (Zhao et al., Biochem BiophysRes Comm 458:790-5, 2015). In vivo, myosin heavy chain protein synthesisrate correlates negatively with TNF-α expression in skeletal muscle, andTNF-α injection into mice induces ubiquitin-proteasome system activationand a decrease of skeletal muscle function (Sakuma K, et al., PflugersArch 467: 213-29, 2015) supporting the mechanism via which an effectiveand well-tolerated anti-inflammatory/TNF-α lowering agent could provideefficacy in sarcopenia and associated disorders involving progressivemuscle loss.

Example 11 Treatment with Thalidomide Analogs

A subject having a condition that may be ameliorated with an embodimentof the disclosed analogs is identified and selected for treatment. Thesubject may have, for example, a disorder mediated by TNF-α activity,TNF-α protein levels, inflammation, and/or angiogenesis. The subject maybe selected based on a clinical presentation and/or by performing teststo demonstrate presence of the disorder.

The subject is treated by administering a thalidomide analog, apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition thereof at an amount determined by a clinician to betherapeutically effective. The compound is administered by any suitablemeans including, but not limited to, orally, parenterally, rectally,nasally, buccally, vaginally, topically, optically, by inhalation spray,or via an implanted reservoir. In some embodiments, the administeredthalidomide analog possesses anti-inflammatory properties. When treatinga disorder that is not mediated by angiogenesis, the thalidomide analogmay not possess anti-angiogenic properties. In certain embodiments, theadministered thalidomide analog is non-teratogenic. Advantageously, thethalidomide analog also may be non-neurotoxic at the administered dose.

A therapeutically effective amount of a second agent may beco-administered with the compound. The compound and the second agent maybe administered either separately or together in a single composition.The second agent may be administered by the same route or a differentroute. If administered concurrently, the compound and the second agentmay be combined in a single pharmaceutical composition or may beadministered concurrently as two pharmaceutical compositions. The secondagent may be, for example, an angiogenesis inhibitor, an anti-canceragent, or an anti-inflammatory agent.

Several representative embodiments are described in the numberedparagraphs below.

1. A thalidomide analog or pharmaceutically acceptable salt thereof,wherein the thalidomide analog is a compound according to generalformula I or

wherein general formula I is

where Y¹ is a bond, —CH₂—, or —CH(CH₃)—; A is —NH₃X where X is an anionwith a −1 charge, or A is general formula II

where bonds represented by “

” are optional bonds, and each bond represented by “

” is a single or double bond as needed to satisfy valence requirements;R¹ is —H, —NO₂, —NH₂, —OC(O)CH₃, or —NO₂H; R² is —H, —NH₂, or—N(H)CH(CH₃)₂; Z¹ is CH₂, C═O, or CH; and Z² is CH₂, C═O, C═S,

Ring B is:

where each bond represented by “

” is a single or double bond as needed to satisfy valence requirements;Z³ is C═O, C═S, or CH; Z⁴ is C═O, C═S, or CH, and at least one of Z³ andZ⁴ is C═O or C═S; R³ is —H or —OH; R⁴ is —H or —CH₃; and R⁵ and R⁶ areboth —H or both —CH₃; wherein:if ring B is

R¹ is —H, —NH₂ or —NO₂, R² and R³ are —H, Z¹ is CH₂, and Z² is C═O, thenY¹ is not a bond;if ring B is

R¹ and R² are H, Z¹ is CH₂ and Z² is C═O, then Y¹ is not a bond;if ring B is

and R¹ and R² are H, then one of Z¹ and Z² is other than C═O; andif ring B is

then (i) R¹ is not —NH₂; (ii) if R¹ is —NO₂ and Y¹ is a bond, then atleast one of Z¹ and Z² is C═O and one of Z³ and Z⁴ is other than C═O orC═S, or if both Z¹ and Z² are C═O, then Z³ is C═O or C═S and Z⁴ is C═S;(iii) if R² is —NH₂ and Y¹ is a bond, then one of Z¹ and Z² is otherthan C═O, and one of Z³ and Z⁴ is other than C═O or C═S; (iv) if R² is—NO₂ and Y¹ is a bond, then one of Z³ and Z⁴ is other than C═O or C═S;(v) if R² is N(H)CH(CH₃)₂, Y¹ is a bond, Z¹ is CH₂ and Z² is C═O, thenone of Z³ and Z⁴ is other than C═O; and (vi) if A is —NH₃X, X is CF₃CO₂⁻, and Y¹ is a bond, then at least one of Z³ and Z⁴ is other than C═O.

2. A method for inhibiting TNF-α activity, angiogenesis, inflammation,or a combination thereof, comprising contacting a cell with an effectiveamount of a thalidomide analog according to paragraph 1, compound 64,compound 72, or compound 77, or a pharmaceutically acceptable saltthereof.

3. The method according to paragraph 2, wherein the thalidomide analogis non-teratogenic.

4. The method according to paragraph 2 or paragraph 3 wherein thethalidomide analog is compound 7, 9, 46, 59, 64, 65, 72, 74, 77, or 86.

5. The method according to paragraph 4, wherein the thalidomide analogis compound 7, 9, 46, 59, 65, 74, or 86.

6. The method according to any one of paragraphs 2-5, wherein contactingthe cell with an effective amount of the thalidomide analog comprisesadministering to a subject a therapeutically effective amount of thethalidomide analog or pharmaceutically acceptable salt thereof or atherapeutically effective amount of a pharmaceutical compositioncomprising the thalidomide analog or pharmaceutically acceptable saltthereof.

7. The method according to paragraph 6, further comprising administeringto the subject a second therapeutic agent.

8. The method according to paragraph 7, wherein the second therapeuticagent is an anti-cancer agent, an anti-angiogenic agent, or ananti-inflammatory agent.

9. A method for inhibiting inflammation in a subject, comprisingadministering to the subject a therapeutically effective amount of athalidomide analog according to paragraph 1, wherein the thalidomideanalog is non-teratogenic and possesses anti-inflammatory properties.

10. The method according to paragraph 9, wherein the thalidomide analogis compound 7, 9, 46, 59, 64, 65, 72, 74, 77, or 86.

11. The method according to paragraph 10, wherein the thalidomide analogis compound 7, 9, 46, 59, 65, 74, or 86.

12. The method according to any one of paragraphs 9-11, wherein thenon-teratogenic compound is administered orally, parenterally, rectally,nasally, buccally, vaginally, topically, optically, by inhalation spray,or via an implanted reservoir.

13. The method according to any one of paragraphs 9-12, furthercomprising administering to the subject a second therapeutic agent.

14. The method of paragraph 13, wherein the second therapeutic agent isan anti-inflammatory agent.

15. A method for treating an inflammatory disorder in a subject,comprising administering to the subject a therapeutically effectiveamount of a thalidomide analog according to paragraph 1, wherein thethalidomide analog is non-teratogenic and possesses anti-inflammatoryproperties.

16. The method according to paragraph 15, wherein the thalidomide analogis compound 7, 9, 46, 59, 64, 65, 72, 74, 77, or 86.

17. The method according to paragraph 16, wherein the thalidomide analogis compound 7, 9, 46, 59, 65, 74, or 86.

18. The method according to any one of paragraphs 15-17, wherein theinflammatory disorder is a neurodegenerative disorder.

19. The method according to paragraph 18, wherein the neurodegenerativedisorder is Alzheimer's disease, Parkinson's disease, head trauma,stroke, amyotrophic lateral sclerosis, human immunodeficiency virusdementia, Huntington's disease, multiple sclerosis, cerebral amyloidangiopathy, a tauopathy, or macular degeneration.

20. The method according to any one of paragraphs 15-19, furthercomprising administering to the subject a second therapeutic agent.

21. The method according to paragraph 20, wherein the second therapeuticagent is an anti-inflammatory agent.

22. A method for inhibiting TNF-α activity in a subject, comprisingadministering to the subject a therapeutically effective amount of athalidomide analog according to paragraph 1, wherein the thalidomideanalog is non-teratogenic.

23. The method according to paragraph 22, wherein the thalidomide analogis compound 7, 9, 46, 59, 64, 65, 72, 74, 77, or 86.

24. The method according to paragraph 23, wherein the thalidomide analogis compound 7, 9, 46, 59, 65, 74, or 86.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A thalidomide analog or pharmaceutically acceptable saltthereof, wherein the thalidomide analog is a compound according togeneral formula I

where Y¹ is a bond, —CH₂—, or —CH(CH₃)—; A is general formula II

where bonds represented by “

” are optional bonds, and each bond represented by “

” is a single or double bond as needed to satisfy valence requirements,R¹ is —H, —NO₂, —NH₂, —OC(O)CH₃, or —NO₂H, R² is —H, —NH₂, or—N(H)CH(CH₃)₂, Z¹ is CH₂, C═O, or CH, and Z² is C═O, CH₂, C═S,

Ring B is:

where each bond represented by “

” is a single or double bond as needed to satisfy valence requirements,Z³ is C═O, C═S, or CH; Z⁴ is C═O, C═S, or CH, and at least one of Z³ andZ⁴ is C═O or C═S, R⁴ is —H or —CH₃ and R⁵ and R⁶ are both —H or both—CH₃; wherein: if ring B is

 then (i) R¹ is not —NH₂, (ii) if R¹ is —NO₂ and Y¹ is a bond, then atleast one of Z¹ and Z² is C═O and one of Z³ and Z⁴ is other than C═O orC═S, or if both Z¹ and Z² are C═O, then Z³ is C═O or C═S and Z⁴ is C═S,(iii) if R² is —NH₂ and Y¹ is a bond, then one of Z¹ and Z² is otherthan C═O, and one of Z³ and Z⁴ is other than C═O or C═S, (iv) if R² is—NO₂ and Y¹ is a bond, then one of Z³ and Z⁴ is other than C═O or C═S,and (v) if R² is N(H)CH(CH₃)₂, Y¹ is a bond, Z¹ is CH₂ and Z² is C═O,then one of Z³ and Z⁴ is other than C═O.
 2. The thalidomide analogaccording to claim 1, wherein ring B is


3. The thalidomide analog according to claim 1, wherein the thalidomideanalog is a compound in Group V, Group I, Group II, or Group III:


4. The thalidomide analog according to claim 3, wherein the thalidomideanalog is compound 7, compound 9, or compound
 44. 5. The thalidomideanalog according to claim 3, wherein the thalidomide analog is compound61, compound 62, or compound
 63. 6. The thalidomide analog according toclaim 3, wherein the thalidomide analog is compound 66, compound 67, orcompound
 68. 7. The thalidomide analog according to claim 1, wherein thethalidomide analog is compound 90, compound 91, compound 92, compound94, compound 96, compound 99, compound 101, compound 103, compound 112,compound 114, compound 115, compound 118, compound 119, compound 120, acompound having a general formula

where ring B is

ring A is

where the bond represented by “

” is a single bond, R¹ is —NO₂, R² is H, Z¹ is H, and Z² is C═O, acompound having a general formula

where ring B is

ring A is

where bonds represented by “

” are absent and each bond represented by “

” is a single bond, R¹ is H, R² is H, Z¹ is H, and Z² is C═O, or acompound having a general formula

where ring B is

ring A is

where bonds represented by “

” are present and each bond represented by “

” is a single bond, R¹ is H, R² is H, Z¹ is C═O, and Z² is C═O.
 8. Apharmaceutical composition comprising a thalidomide analog orpharmaceutically acceptable salt thereof according to claim 1 and atleast one pharmaceutically acceptable carrier.
 9. A method forinhibiting TNF-α activity, TNF-α synthesis, angiogenesis, inflammation,or a combination thereof, comprising contacting a cell with an effectiveamount of a thalidomide analog according to claim
 1. 10. The methodaccording to claim 9, wherein the thalidomide analog is compound 7,compound 9, compound 44, compound 61, compound 62, compound 63, compound66, compound 67, compound 68, compound 90, compound 91, compound 92,compound 94, compound 96, compound 99, compound 101, compound 103,compound 112, compound 114, compound 115, compound 118, compound 119,compound 120, a compound having a general formula

where ring B is

ring A is

where the bond represented by “

” is a single bond, R¹ is —NO₂, R² is H, Z¹ is H, and Z² is C═O, acompound having a general formula

where ring B is

ring A is

where bonds represented by “

” are absent and each bond represented by “

” is a single bond, R¹ is H, R² is H, Z¹ is H, and Z² is C═O, a compoundhaving a general formula

where ring B is

ring A is

where bonds represented by “

” are present and each bond represented by “

” is a single bond, R¹ is H, R² is H, Z¹ is C═O, and Z² is C═O, or anycombination thereof.
 11. The method according to claim 9, whereincontacting the cell with an effective amount of the thalidomide analogcomprises administering to a subject a therapeutically effective amountof the thalidomide analog or a pharmaceutically acceptable salt thereofor a therapeutically effective amount of a pharmaceutical compositioncomprising the thalidomide analog or a pharmaceutically acceptable saltthereof.
 12. The method according to claim 11, wherein the thalidomideanalog is compound 7, compound 9, compound 44, compound 61, compound 62,compound 63, compound 66, compound 67, compound 68, compound 90,compound 91, compound 92, compound 94, compound 96, compound 99,compound 101, compound 103, compound 112, compound 114, compound 115,compound 118, compound 119, compound 120, a compound having a generalformula

where ring B is

ring A is

where the bond represented by “

” is a single bond, R¹ is —NO₂, R² is H, Z¹ is H, and Z² is C═O, acompound having a general formula

where ring B is

ring A is

where bonds represented by “

” are absent and each bond represented by “

” is a single bond, R¹ is H, R² is H, Z¹ is H, and Z² is C═O, a compoundhaving a general formula

where ring B is

ring A is

where bonds represented by “

” are present and each bond represented by “

” is a single bond, R¹ is H, R² is H, Z¹ is C═O, and Z² is C═O, or anycombination thereof.
 13. The method according to claim 11 wherein thesubject has an inflammatory disorder or an autoimmune disorder.
 14. Themethod according to claim 13, wherein the thalidomide analog possessesanti-inflammatory properties, and administering the therapeuticallyeffective amount of the thalidomide analog or pharmaceuticallyacceptable salt thereof or the therapeutically effective amount of thepharmaceutical composition inhibits inflammation in the subject.
 15. Themethod according to claim 12, wherein the subject has an inflammatorydisorder, the thalidomide analog possesses anti-inflammatory properties,and administering the therapeutically effective amount of thethalidomide analog or pharmaceutically acceptable salt thereof or thetherapeutically effective amount of the pharmaceutical composition tothe subject treats the inflammatory disorder.
 16. The method accordingto claim 11, wherein administering the therapeutically effective amountof the thalidomide analog or pharmaceutically acceptable salt thereof orthe therapeutically effective amount of the pharmaceutical compositionto the subject inhibits TNF-α activity, TNF-α synthesis, or acombination thereof in the subject.
 17. The method according to claim11, wherein the thalidomide analog possesses anti-angiogenic properties,and administering the therapeutically effective amount of thethalidomide analog or pharmaceutically acceptable salt thereof or thetherapeutically effective amount of the pharmaceutical composition tothe subject inhibits angiogenesis in the subject.
 18. The methodaccording to claim 11, wherein the thalidomide analog orpharmaceutically acceptable salt thereof or the pharmaceuticalcomposition is administered orally, parenterally, rectally, nasally,buccally, vaginally, topically, optically, by inhalation spray, or viaan implanted reservoir.
 19. The method according to claim 11, furthercomprising administering to the subject a second therapeutic agent. 20.The method according to claim 19, wherein the second therapeutic agentis an anti-cancer agent, an anti-angiogenic agent, or ananti-inflammatory agent.